Compositions and methods for identifying antigens which elicit an immune response

ABSTRACT

This invention relates to an expression vector wherein said expression vector comprises a polynucleotide promoter sequence, a polynucleotide encoding a signal sequence, a polynucleotide encoding an antigen protein or peptide, a polynucleotide encoding a cell binding element, and a polynucleotide polyadenylation sequence all operatively linked. More particularly, it relates to the method of eliciting an immune response directed against an antigen in a mammal comprising the steps of introducing the expression vector into a cell, expressing the vector to produce an antigen under conditions wherein the antigen is secreted from the cell, endocytosing the secreted antigen into the cell, processing the antigen, and presenting fragments to a receptor to elicit a T-cell response. In addition, this invention relates to a vaccine and a method of use. The invention also relates to the method of identifying MHC-II restricted epitopes.

This application is a continuation application of prior application Ser.No. 09/566,420, filed May 5, 2000, now allowed, which claims priority toU.S. Provisional Application No. 60/132,752, filed May 6, 1999 and U.S.Provisional Application No. 60/132,750, filed May 6, 1999.

This invention was made using finds obtained from the U.S. Government(National Institutes of Health Grant No. RO1 A1419595-01) and the U.S.Government may therefore have certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to an expression vector and its use to elicit acomplete immune response in a mammal. More particularly it relates theprocessing of an endogenous antigen as an exogenous antigen forpresentation on MHC-II. This invention also relates to a vaccine and itsmethod of use to immunize a mammal.

BACKGROUND OF THE INVENTION

Inadequate antigen presentation in humans results in the failure ofhuman immune system to control and clear many pathogenic infections andmalignant cell growth. Successful therapeutic vaccines andimmunotherapies for chronic infection and cancer rely on the developmentof new approaches for efficient antigen presentation to induce avigorous immune response which is capable of controlling and clearingthe offensive antigens.

The ability of T cells to recognize an antigen is dependent onassociation of the antigen with either MHC Class I (MHC-I) or Class II(MCH-II) proteins. For example, cytotoxic T cells respond to an antigenin association with MHC-I proteins. Thus, a cytotoxic T cell that killsa virus-infected cell will not kill a cell infected with the same virusif the cell does not also express the appropriate MHC-I protein. HelperT cells recognize MHC-II proteins. Helper T cell activity depends ingeneral on both the recognition of the antigen on antigen presentingcells and the presence on these cells of “self” MHC-II proteins. Thisrequirement to recognize an antigen in association with a self-MHCprotein is called MHC restriction. MHC-I proteins are found on thesurface of virtually all nucleated cells. MHC-II proteins are found onthe surface of certain cells including macrophages, B cells, anddendritic cells of the spleen and Langerhans cells of the skin.

A crucial step in mounting an immune response in mammals, is theactivation of CD4+ helper T-cells that recognize majorhistocompatibility complexes (MHC)-II restricted exogenous antigens.These antigens are captured and processed in the cellular endosomalpathway in antigen presenting cells, such as dendritic cells (DCs)(Zajac et al., 1998; Bona et al., 1998; Kalams et al., 1998; Mellman etal., 1998; Banchereau et al., 1998). In the endosome and lysosome, theantigen is processed into small antigenic peptides that are presentedonto the MHC-II in the Golgi compartment to form an antigen-MHC-IIcomplex. This complex is expressed on the cell surface, which expressioninduces the activation of CD4+ T cells.

Other crucial events in the induction of an effective immune response inan animal involve the activation of CD8+ T-cells and B cells. CD8+ cellsare activated when the desired protein is routed through the cell insuch a manner so as to be presented on the cell surface as processedproteins, which are complexed with MHC-I antigens. B cells can interactwith the antigen via their surface immunoglobulins (IgM and IgD) withoutthe need for MHC proteins. However, the activation of the CD4+ T-cellsstimulates all arms of the immune system. Upon activation, CD4+ T-cells(helper T cells) produce interleukins. These interleukins help activatethe other arms of the immune system. For example, helper T cells produceinterleukin-4 (IL-4) and interleukin-5 (IL-5), which help B cellsproduce antibodies; interleukin-2 (IL-2), which activates CD4+ and CD8+T-cells; and gamma interferon, which activates macrophages.

Since helper T-cells that recognize MHC-II restricted antigens play acentral role in the activation and clonal expansion of cytotoxicT-cells, macrophages, natural killer cells and B cells, the initialevent of activating the helper T cells in response to an antigen iscrucial for the induction of an effective immune response directedagainst that antigen. Attempts to stimulate helper T-cell activationusing a sequence derived from the lysosomal transmembrane proteins havebeen reported (Wu, 1995). However, these attempts did not result in theinduction of effective immune responses with respect to CD8+ T-cells andB cells in the mammals being tested.

Thus, there is a long felt need in the art for efficient and directedmeans of eliciting an immune response for the treatment of diseases inmammals. The present invention satisfies this need.

SUMMARY OF THE INVENTION

An embodiment of the present invention is an expression vectorcomprising a polynucleotide promoter sequence, a polynucleotide encodinga signal sequence, a polynucleotide encoding an antigen, apolynucleotide encoding a cell binding element, and a polynucleotidepolyadenylation sequence all operatively linked.

In specific embodiments of the present invention, the polynucleotidepromoter sequence is selected from the group consisting of aconstitutive promoter, an inducible promoter and a tissue specificpromoter.

In another specific embodiment of the present invention, thepolynucleotide encoding a signal sequence is selected from the groupconsisting of a hepatitis B virus E antigen signal sequence, animmunoglobulin heavy chain leader sequence, and a cytokine leadersequence.

An embodiment of the present invention is an expression vector whereinthe polynucleotide encoding an antigen comprises a polynucleotidesequence for at least one epitope, wherein said at least one epitopeinduces a B cell response in a mammal.

A further embodiment of the present invention is an expression vectorwherein the polynucleotide encoding an antigen comprises apolynucleotide sequence for at least one epitope, wherein said at leastone epitope induces a CD4+ T-cell response in a mammal.

Another embodiment of the present invention is an expression vectorwherein the polynucleotide encoding an antigen comprises apolynucleotide sequence for at least one epitope, wherein said at leastone epitope induces a CD8+ T-cell response in a mammal.

A specific embodiment of the present invention is an expression vectorwherein the polynucleotide sequence encoding an antigen comprises apolynucleotide sequence for at least one epitope, wherein said at leastone epitope induces a B cell response, a CD4+ T-cell response and a CD8+T-cell response in a mammal into which said antigen is introduced.

A further specific embodiment of the present invention is an expressionvector wherein the polynucleotide sequence encoding an antigen comprisesa polynucleotide sequence for a plurality of epitopes, wherein saidplurality of epitopes induces a B cell response, a CD4+ T-cell responseand a CD8+ T-cell response in a mammal into which said antigen isintroduced.

A further embodiment of the present invention is an expression vectorwherein the polynucleotide encoding a cell binding element is apolynucleotide sequence of a ligand which binds to a cell surfacereceptor. In specific embodiments, the cell binding element sequence isselected from the group consisting of polynucleotide sequences whichencode a Fc fragment, a toxin cell binding domain, a cytokine, a smallpeptide and an antibody. In specific embodiments, the polynucleotideencoding a cell binding element is a homologous polynucleotide sequenceor a heterologous polynucleotide sequence.

An additional embodiment of the present invention is a transformed cellcomprising an expression vector wherein said expression vector comprisesa polynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding an antigen, a polynucleotideencoding a cell binding element, and a polynucleotide polyadenylationsequence all operatively linked.

Another specific embodiment of the present invention is a fusion proteinwherein the fusion protein comprises a signal sequence, an antigen and acell binding element. In specific embodiments, antigen presenting cellshave been transduced with the fusion protein in vitro. (In furtherembodiments, the fusion protein is administered directly to a mammal.

A specific embodiment of the present invention is a vaccine comprisingan expression vector wherein said expression vector comprises apolynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding an antigen, a polynucleotideencoding a cell binding element, and a polynucleotide polyadenylationsequence all operatively linked. In specific embodiments, a vaccinecomprises antigen presenting cells, wherein said antigen presentingcells are transduced in vitro with the expression vector. In furtherembodiments, a vaccine comprises antigen presenting cells, wherein saidantigen presenting cells are transduced in vitro with the fusionprotein.

Another specific embodiment of the present invention is an expressionvector comprising at least a polynucleotide encoding a signal sequence,a polynucleotide encoding an antigen and a polynucleotide encoding acell binding element.

A further embodiment of the present invention is a method to elicit animmune response directed against an antigen, comprising the steps of:introducing an expression vector into a cell, wherein said expressionvector comprises a polynucleotide promoter sequence, a polynucleotideencoding a signal sequence, a polynucleotide encoding an antigen, apolynucleotide encoding a cell binding element, and a polynucleotidepolyadenylation sequence, all operatively linked; and expressing saidvector to produce an antigen under conditions wherein said antigen issecreted from the cell; said secreted antigen is endocytosed into thecell; said endocytosed antigen is processed inside the cell; and saidprocessed antigen is presented to a cell surface protein, to elicit aT-cell mediated immune response. In specific embodiments, the antigen issecreted by a first cell and internalized by a second cell wherein thefirst and second cells are antigen presenting cells. In furtherembodiments, the first cells is a non-antigen presenting cell and thesecond cell is an antigen presenting cell.

Another specific embodiment of the present invention is a method toidentify a polynucleotide sequence which encodes at least one MHC-IIrestricted epitope that is capable of activating CD4+ helper T-cells,said method comprising the steps of: introducing an expression vectorinto an antigen presenting cell to produce a transduced antigenpresenting cell, wherein said expression vector comprises apolynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding a test polypeptide, a polynucleotideencoding a cell binding element, and a polynucleotide polyadenylationsequence, all operatively linked; contacting said transduced antigenpresenting cell with naive or primed T-cells; and assessing whether anynaive T-cells or primed T-cells are activated upon contact with saidtransduced antigen presenting cell, wherein activation of any of saidT-cells indicates that the polynucleotide encoding the test polypeptideis a gene or fragment thereof capable of activating CD4+ helper T-cells.In specific embodiments, the polynucleotide encoding a test polypeptideis selected from the group of cDNA libraries consisting of viralgenomes, bacterial genomes, parasitic genomes and human genomes.

Another embodiment of the present invention is a method to identify apolynucleotide sequence which encodes at least one MHC-II restrictedepitope that is capable of eliciting an immune response in vivo, saidmethod comprising the steps of: introducing an expression vector intoantigen presenting cells to produce transduced antigen presenting cells,wherein said expression vector comprises a polynucleotide promotersequence, a polynucleotide encoding a signal sequence, a polynucleotideencoding a test polypeptide, a polynucleotide encoding a cell bindingelement, and a polynucleotide polyadenylation sequence, all operativelylinked; administering said transduced antigen presenting cells to amammal via a parenteral route; collecting T-cells from splenocytes andco-culturing with dendritic cells; and assessing activation of T-cells,wherein said activation of T-cells indicate that the polynucleotideencoding the test polypeptide is a gene or fragment thereof capable ofactivating CD4+ helper T-cells. In specific embodiments, thepolynucleotide encoding a test polypeptide is selected from the group ofcDNA libraries consisting of viral genomes, bacterial genomes, parasiticgenomes and human genomes.

A specific embodiment of the present invention is a method to identify apolynucleotide sequence which encodes at least one MHC-II restrictedepitope that is capable of eliciting an immune response in vivo, saidmethod comprising the steps of: administering to a mammal via parenteralroute an expression vector, wherein said expression vector comprises apolynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding a test polypeptide, a polynucleotideencoding a cell binding element, and a polynucleotide polyadenylationsequence, all operatively linked; administering said transduced antigenpresenting cells to a mammal via a parenteral route; collecting T-cellsfrom splenocytes and co-culturing with dendritic cells; and assessingactivation of T-cells, wherein said activation of T-cells indicate thatthe polynucleotide encoding the test polypeptide is a gene or fragmentthereof capable of activating CD4+ helper T-cells. In specificembodiments, the polynucleotide encoding a test polypeptide is selectedfrom the group of cDNA libraries consisting of viral genomes, bacterialgenomes, parasitic genomes and human genomes.

A specific embodiment of the present invention is a method of treatingcancer comprising the steps of identifying a test polypeptide whichencodes at least one MHC-II restricted epitope, wherein said polypeptideis identified under the conditions of transducing antigen presentingcells with an expression vector into antigen presenting cells to producetransduced antigen presenting cells, wherein said expression vectorcomprises a polynucleotide promoter sequence, a polynucleotide encodinga signal sequence, a polynucleotide encoding a test polypeptide, apolynucleotide encoding a cell binding element, and a polynucleotidepolyadenylation sequence, all operatively linked and assessingactivation of T-cells, wherein said activation of T-cells indicate thatthe polynucleotide encoding the test polypeptide is a gene or fragmentthereof capable of activating CD4+ helper T-cells; and administeringantigen presenting cells to a mammal via a parenteral route, whereinsaid antigen presenting cells are transduced with the test polypeptide.

Another specific embodiment of the present invention is a method oftreating cancer comprising the steps of identifying a test polypeptidewhich encodes at least one MHC-II restricted epitope, wherein saidpolypeptide is identified under the conditions of transducing antigenpresenting cells with an expression vector into antigen presenting cellsto produce transduced antigen presenting cells, wherein said expressionvector comprises a polynucleotide promoter sequence, a polynucleotideencoding a signal sequence, a polynucleotide encoding a testpolypeptide, a polynucleotide encoding a cell binding element, and apolynucleotide polyadenylation sequence, all operatively linked andassessing activation of T-cells, wherein said activation of T-cellsindicate that the polynucleotide encoding the test polypeptide is a geneor fragment thereof capable of activating CD4+ helper T-cells; andadministering to a mammal via a parenteral route an expression vector,wherein said expression vector comprises at least the polynucleotideencoding the test polypeptide and a polynucleotide encoding a cellbinding element said antigen presenting cells are transduced with thetest polypeptide.

A further specific embodiment of the present invention is a method oftreating a viral infection comprising the steps of identifying a testpolypeptide which encodes at least one MHC-II restricted epitope,wherein said polypeptide is identified under the conditions oftransducing antigen presenting cells with an expression vector intoantigen presenting cells to produce transduced antigen presenting cells,wherein said expression vector comprises a polynucleotide promotersequence, a polynucleotide encoding a signal sequence, a polynucleotideencoding a test polypeptide, a polynucleotide encoding a cell bindingelement, and a polynucleotide polyadenylation sequence, all operativelylinked and assessing activation of T-cells, wherein said activation ofT-cells indicate that the polynucleotide encoding the test polypeptideis a gene or fragment thereof capable of activating CD4+ helper T-cells;and administering antigen presenting cells to a mammal via a parenteralroute, wherein said antigen presenting cells are transduced with thetest polypeptide.

Another embodiment of the present invention is a method of treating aviral infection comprising the steps of identifying a test polypeptidewhich encodes at least one MHC-II restricted epitope, wherein saidpolypeptide is identified under the conditions of transducing antigenpresenting cells with an expression vector into antigen presenting cellsto produce transduced antigen presenting cells, wherein said expressionvector comprises a polynucleotide promoter sequence, a polynucleotideencoding a signal sequence, a polynucleotide encoding a testpolypeptide, a polynucleotide encoding a cell binding element, and apolynucleotide polyadenylation sequence, all operatively linked andassessing activation of T-cells, wherein said activation of T-cellsindicate that the polynucleotide encoding the test polypeptide is a geneor fragment thereof capable of activating CD4+ helper T-cells; andadministering to a mammal via a parenteral route an expression vector,wherein said expression vector comprises at least the polynucleotideencoding the test polypeptide and a polynucleotide encoding a cellbinding element said antigen presenting cells are transduced with thetest polypeptide.

Another embodiment of the present invention is a method of treating, anautoimmune disease comprising the steps of identifying a testpolypeptide which encodes at least one MHC-II restricted epitope,wherein said polypeptide is identified under the conditions oftransducing antigen presenting cells with an expression vector intoantigen presenting cells to produce transduced antigen presenting cells,wherein said expression vector comprises a polynucleotide promotersequence, a polynucleotide encoding a signal sequence, a polynucleotideencoding a test polypeptide, a polynucleotide encoding a cell bindingelement, and a polynucleotide polyadenylation sequence, all operativelylinked and assessing activation of T-cells, wherein said activation ofT-cells indicate that the polynucleotide encoding the test polypeptideis a gene or fragment thereof capable of activating CD4+ helper T-cells;and administering antigen presenting cells to a mammal via a parenteralroute, wherein said antigen presenting cells are transduced with thetest polypeptide.

A specific embodiment of the present invention is a method of treatingan autoimmune disease comprising the steps of identifying a testpolypeptide which encodes at least one MHC-II restricted epitope,wherein said polypeptide is identified under the conditions oftransducing antigen presenting cells with an expression vector intoantigen presenting cells to produce transduced antigen presenting cells,wherein said expression vector comprises a polynucleotide promotersequence, a polynucleotide encoding a signal sequence, a polynucleotideencoding a test polypeptide, a polynucleotide encoding a cell bindingelement, and a polynucleotide polyadenylation sequence, all operativelylinked and assessing activation of T-cells, wherein said activation ofT-cells indicate that the polynucleotide encoding the test polypeptideis a gene or fragment thereof capable of activating CD4+ helper T-cells;and administering to a mammal via a parenteral route an expressionvector, wherein said expression vector comprises at least thepolynucleotide encoding the test polypeptide and a polynucleotideencoding a cell binding element said antigen presenting cells aretransduced with the test polypeptide.

A further embodiment of the present invention is a method of producing avaccine to immunize a mammal comprising the steps of: transducingantigen presenting cell by introducing an expression vector into a cell,wherein said expression vector comprises a polynucleotide promotersequence, a polynucleotide encoding a signal sequence, a polynucleotideencoding an antigen, a polynucleotide encoding a cell binding element,and a polynucleotide polyadenylation sequence, all operatively linked;and expressing said vector to produce an antigen under conditionswherein said antigen is secreted from the cell. In specific embodiments,antigen presenting cells are transduced with the antigen in vitro or exvivo prior to administering the antigen presenting cells to the mammal.

Another specific embodiment of the present invention is a method ofinducing an immune response comprising the steps of co-administering toa mammal a cytokine expression vector and a retrogen expression vector,wherein the retrogen expression vector comprises a polynucleotidepromoter sequence, a polynucleotide encoding a signal sequence, apolynucleotide encoding an antigen, a polynucleotide encoding a cellbinding element, and a polynucleotide polyadenylation sequence alloperatively linked.

A further embodiment of the present invention is a method of inducing animmune response comprising the steps of co-administering to a mammal oneexpression vector, wherein said expression vector comprises apolynucleotide sequence encoding a cytokine protein and a polynucleotidesequence encoding a fusion protein under transcriptional control of onepromoter, wherein said fusion protein comprises an antigen and a cellbinding element. In specific embodiments, the polynucleotide sequenceencoding the cytokine protein and the polynucleotide sequence encodingthe fusion protein are under separate transcriptional control, andwherein the polynucleotide sequence encoding the cytokine protein andthe polynucleotide sequence encoding the fusion protein are in tandem inthe one expression vector.

Another embodiment of the present invention is a method of inducing animmune response comprising the steps of co-administering to a mammal twodifferent retrogen expression vectors, wherein a first retrogenexpression vector comprises a polynucleotide promoter sequence, apolynucleotide encoding a signal sequence, a polynucleotide encoding afirst antigen, a polynucleotide encoding a cell binding element, and apolynucleotide polyadenylation sequence all operatively linked; and asecond retrogen expression vector comprises a polynucleotide promotersequence, a polynucleotide encoding a signal sequence, a polynucleotideencoding a second antigen, a polynucleotide encoding a cell bindingelement, and a polynucleotide polyadenylation sequence all operatively.

Another specific embodiment of the present invention is a method ofinducing an immune response comprising the steps of administering to amammal one expression vector, wherein said expression vector comprises apolynucleotide sequence encoding a first fusion protein and apolynucleotide sequence encoding a second fusion protein undertranscriptional control of one promoter, wherein said first fusionprotein comprises a first antigen and a first cell binding element andsaid second fusion protein comprises a second antigen and a first cellbinding element. In specific embodiments, the first and second antigensare different antigens and the cell binding elements is a Fc fragment.In further embodiments, the first and second antigens are differentantigens and the first and second cell binding elements are differentcell binding elements. An additional embodiment includes that thepolynucleotide sequence encoding the first fusion protein and thepolynucleotide sequence encoding the second fusion protein are underseparate transcriptional control, and wherein the polynucleotidesequence encoding the first fusion protein and the polynucleotidesequence encoding the second fusion protein are in tandem in oneexpression vector.

A specific embodiment of the present invention is a method ofsimultaneously inducing both CD4+ and CD8+ T-cells comprising the stepsof administering a fusion protein wherein the protein comprises both aMHC-I and MHC-II epitope fused to a cell binding element.

A further embodiment of the present invention is a method of producing afusion protein comprising the steps of introducing an expression vectorinto a cell, wherein said expression vector comprises a polynucleotidepromoter sequence, a polynucleotide encoding a signal sequence, apolynucleotide encoding an antigen, a polynucleotide encoding a cellbinding element, and a polynucleotide polyadenylation sequence, alloperatively linked and expressing said vector to produce a fusionprotein under conditions wherein said fusion protein is secreted fromthe cell. In specific embodiments, antigen presenting cells aretransduced with the fusion protein in vitro.

A specific embodiment of the present invention is a method of secretingan intracellular protein comprising the steps of introducing anexpression vector into a cell, wherein said expression vector comprisesa polynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding an intracellular protein, apolynucleotide encoding a cell binding element, and a polynucleotidepolyadenylation sequence, all operatively linked and expressing saidvector to produce a fusion protein under conditions wherein said fusionprotein is secreted from the cell. More specifically, the polynucleotidesequence encoding the intracellular protein is truncated or mutated toincrease efficiency of secretion.

Another specific embodiment of the present invention is a method ofsecreting a membrane protein comprising the steps of introducing anexpression vector into a cell, wherein said expression vector comprisesa polynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding a membrane protein, a polynucleotideencoding a cell binding element, and a polynucleotide polyadenylationsequence, all operatively linked and expressing said vector to produce afusion protein under conditions wherein said fusion protein is secretedfrom the cell. More specifically, the polynucleotide sequence encodingthe membrane protein is truncated or mutated to increase efficiency ofsecretion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B are diagrams representing the retrogen strategy ofthe invention. The retrogen of the invention is produced in a cell, forexample, a muscle cell (FIG. 1A), and is then taken up by an antigenpresenting cell (FIG. 1B). The retrogen is processed in the antigenpresenting cell and is expressed thereon as a MHC-I or a MHC-II complex,or presented to B cell receptors as shown in the FIG. 1A and FIG. 1B.MHC-I presentation of the retrogen results in the activation ofcytotoxic CD8+ T-cells and MHC-II presentation of the retrogen resultsin the activation of CD4+ T-cells.

FIG. 2A, FIG. 2B and FIG. 2C are a series of schematic representationsof the expression vectors. FIG. 2A illustrates a vector comprising HBeAg(secretory), HBcAg (cytosolic), or the Fc fragment with a signalsequence (secretory) constructed by generating a fusion gene as shown inthe diagram, and cloning the gene into the retroviral vector (LNC-NGFR)or the expression vector pRc/CMV. FIG. 2B and FIG. 2C illustrateadditional vectors that were constructed.

FIG. 3 is an image of a Western blot depicting expression and secretionof the HBe-retrogen. COS cells were transfected with various expressionvectors. The culture medium (M) and cell lysates (C) were thenprecipitated with an anti-IgG or anti-HbeAg antibody and analyzed bySDS-PAGE.

FIG. 4A, FIG. 4B and FIG. 4C are a series of graphs depictingtransduction and expression of retrogen in dendritic cells. Murine bonemarrow cells were transduced with various recombinant retroviralvectors; the cells were matured into dendritic cells in the presence ofGM-CSF, TNF, and IL-4 and stained with the anti-NGFR. They are measuredby a flow cytometric assay. FIG. 4A shows the untransduced dendriticcells. FIG. 4B shows the transduced dendritic cells. FIG. 4C is anegative control.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D and FIG. 5E are a series of graphsdepicting the presence of surface markers (MHC-I, MHC-II,co-stimulation, and adhesion molecules (CD11C, CD54, CD80 and CD86)) ondendritic cells as determined by flow cytometric assays. FIG. 5A showsthe presence of CD11C surface marker. FIG. 5B shows the presence of CD54surface marker. FIG. 5C shows the presence of CD80 surface marker. FIG.5D shows the presence of CD86 surface marker. And FIG. 5E shows thepresence of MHC-II.

FIG. 6A and FIG. 6B illustrate two bar graphs depicting in vitroactivation of naive CD4+ T-cells by retrogen-transduced dendritic cells.FIG. 6A shows levels of GM-CSF in co-culture. FIG. 6B shows the levelsof IFN-γ in the co-culture medium.

FIG. 7A and FIG. 7B illustrate the MHC-II-dependent activation. FIG. 7Ashows the cytokine concentration (IFN-γ) from cells obtained fromMHC-II-knockout (KO) or wild-type (WT) C57BL/6 mice transduced with theHBe-retrogen and co-cultured and naive CD4+ T-cells from wild type mice.FIG. 7B shows the GM-CSF cytokine concentration.

FIG. 8 shows antibody responses in the sera of immunized mice.

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E and FIG. 9F show theconstruction and expression of s-MAGE-3-Fc fusion proteins. FIG. 9Ashows the schematic representation of recombinant retroviral vectors.(S: the signal sequence. IRES: Internal ribosome entry site sequence.)FIG. 9B shows the expression of different constructs in dendritic cellsas determined by Western blot analysis stained with the mouseanti-MAGE-3 and an anti-mouse IgG HRP conjugate. FIG. 9C shows theprotein band intensity of the Western blot of FIG. 9B analyzed by aPhosphorImager (Molecular Dynamics) with an Image-Quant software. FIG.9D, FIG. 9E and FIG. 9F illustrate the flow cytometric analysis oftransduced dendritic cells transduced with each construct and stainedfor MHC-II (FIG. 9E) (M5/114.15.2), CD40 (FIG. 9D) (HM40-3), andCD86/B7.2 (FIG. 9F) (GL1) (PharMingen).

FIG. 10A, FIG. 10B, FIG. 10C and FIG. 10D show the in vivo induction ofCD4+ Th1 responses of mice immunized with dendritic cells transducedwith different vectors in the media after co-culture of CD4+ T-cells.FIG. 10A shows the concentrations of IFN-γ. FIG. 10B shows theconcentrations of IL-2. FIG. 10C shows the concentrations of TNF-α. FIG.10D shows the concentrations of IL-4.

FIG. 11A and FIG. 11B show the IFN-γ levels in CD4+ T-cells isolatedfrom s-MAGE-3-Fc-dendritic cells immunized mice co-cultured withs-MAGE-3-Fc-dendritic cells in the presence or absence of anti-CD4 oranti-CD8 antibodies (FIG. 11A), or co-cultured with HBcAg transduceddendritic cells (FIG. 11B).

FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D show the cytokine levels inCD4+ T-cells isolated from pooled splenocytes of mice immunized withdendritic cells co-cultured with dendritic cells isolated from draininglymph nodes (LN) of the same immunized mice at a ratio of 1000:1. FIG.12A shows the concentrations of IFN-γ. FIG. 12B shows the concentrationsof IL-2. FIG. 12C shows the concentrations of TNF-α. FIG. 12D shows theconcentrations of IL-4.

FIG. 13 shows the in vivo induction of cytotoxicity responses fromsplenocytes isolated from immunized mice which were re-stimulated (E) invitro with irradiated EL4-MAGE-3 cells and co-cultured with the³H-thymidine labeled target cells, EL4-MAGE-3 or EL4-HBcAg (control)(T).

FIG. 14 shows the induction of antibody responses 6 weeks afterdendritic cell immunization.

FIG. 15 shows the enhanced interaction of T-cells withs-MAGE-3-Fc-dendritic cells by measuring the IL-12 levels in theco-culture in the presence or absence of an anti-CD40L antibody (MR1,PharMingen) measured by ELISA.

FIG. 16A and FIG. 16B show the antitumor immunity of mice that wereimmunized by i.v. injection with 1×10⁵ dendritic cells transduced withdifferent constructs before inoculated intradermally inoculatedEL4-MAGE-3 tumor cells. FIG. 16A shows the tumor volumes. FIG. 16B showsthe percentage of surviving mice in each group.

FIG. 17 illustrates the charged amino acid residues of HPV 16E7, whichwere deleted to stabilize the protein and facilitate secretion.

FIG. 18 illustrates a schematic representation of expression vectors.The HBe-Fc fusion gene, HBcAg (cytosolic) gene, HBeAg (secretory) gene,or Fc cDNA fragment with a signal sequence (secretory) was cloned intothe pRc/CMV vector under the CMV promoter control, respectively. Theblack square represents the signal sequence.

FIG. 19A and FIG. 19B show the expression of HBe-Fc, HBcAg, HBeAg, andFc constructs. FIG. 19A shows the expression of the different constructsexpressed in cells as determined by Western blot analysis. FIG. 19Bshows the protein band intensity of the Western blot in FIG. 19Aanalyzed by a PhosphoImager (Molecular Dynamics) with an Image-Quantsoftware.

FIG. 20 illustrates the in vivo induction of T-cell responses of miceafter DNA immunization with different plasmids or primed T cells thatwere sacrificed 4 weeks after immunization. Splenocytes werere-stimulated by HBe/cAg recombinant proteins for 5 days.

FIG. 21A, FIG. 21B, FIG. 21C and FIG. 21D illustrate the in vivoinduction of CD4+ T-cell responses of mice that were immunized withdifferent plasmids and sacrificed 4 weeks after immunization. FIG. 21Aand FIG. 21B show CD4+ T cells that were co-cultured in duplicate withHBe/cAg pulsed-dendritic cells. FIG. 21C and FIG. 21D show CD4+ T cellsfrom the HBeFc immunized mice that were co-cultured with HBe/cAgpulsed-dendritic cells in the presence or absence of anti-CD4+ oranti-CD8+ antibodies. The concentrations of IFN-γ and IL-2 in the mediawere determined by ELISA after 72 hours of co-culture.

FIG. 22 illustrates the in vivo induction of CTL responses insplenocytes that were isolated from DNA immunized mice and restimulatedin vitro with irradiated EL4-HBcAg cells for 5 days. The restimulatedsplenocytes (E) were co-cultured for 4 hr with the ³H-labeled targetcells, EL4-HbcAg or EL4-MAGE3 (control) (T).

FIG. 23 shows the induction of antibody responses. The HBc/eAg-specificIgG antibodies from mice at 4-6 weeks after DNA immunization weredetermined by ELISA.

FIG. 24 illustrates data from the dendritic cell transfer experiment.The CD11c+ dendritic cells were isolated from the splenocytes of donormice immunized with DNA vaccines. The primed-dendritic cells wereinjected into the lateral tail vein of syngeneic naive recipients. Twoto four weeks after the adoptive transfer, T-cell proliferation assayswere performed.

FIG. 25 illustrates a schematic of retroviral vectors for theconstruction of cDNA libraries to identify MHC-II restricted epitopes.

FIG. 26 illustrates a schematic of the process to identify MHC-IIrestricted epitopes capable of eliciting a CD4+ helper T-cell response.

DETAILED DESCRIPTION

It is readily apparent to one skilled in the art that variousembodiments and modifications may be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

The term “antibody” as used herein, refers to an immunoglobulinmolecule, which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoactive portions ofintact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1988; Houstonet al., 1988; Bird et al., 1988).

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. An antigen can be derived from organisms, subunits ofproteins/antigens, killed or inactivated whole cells or lysates.Exemplary organisms include but are not limited to, Helicobacters,Campylobacters, Clostridia, Corynebacterium diphtheriae, Bordetellapertussis, influenza virus, parainfluenza viruses, respiratory syncytialvirus, Borrelia burgdorfei, Plasmodium, herpes simplex viruses, humanimmunodeficiency virus, papillomavirus, Vibrio cholera, E. coli, measlesvirus, rotavirus, shigella, Salmonella typhi, Neisseria gonorrhea.Therefore, a skilled artisan realizes that any macromolecule, includingvirtually all proteins or peptides, can serve as antigens. Furthermore,antigens can be derived from recombinant or genomic DNA. A skilledartisan realizes that any DNA, which contains nucleotide sequences orpartial nucleotide sequences of a pathogenic genome or a gene or afragment of a gene for a protein that elicits an immune response resultsin synthesis of an antigen. Furthermore, one skilled in the art realizesthat the present invention is not limited to the use of the entirenucleic acid sequence of a gene or genome. It is readily inherent thatthe present invention includes, but is not limited to, the use ofpartial nucleic acid sequences of more than one gene or genome and thatthese nucleic acid sequences are arranged in various combinations toelicit the desired immune response.

The term “autoimmune disease” as used herein is defined as a disorderthat results from autoimmune responses. Autoimmunity is an inappropriateand excessive response to self-antigens. Examples include but are notlimited to, Addision's disease, Graves' disease, Type I-Diabetesmellitus, Multiple sclerosis, Myxedema, Pernicious anemia, Rheumaticfever, Rheumatoid arthritis, Systemic lupus erythematous, and ulcerativecolitis.

The term “cancer” as used herein is defined a proliferation of cellswhose unique trait—loss of normal controls—results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis.Examples include but are not limited to, breast cancer, prostate cancer,ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,colorectal cancer, renal cancer and lung cancer.

The terms “cell,” “cell line,” and “cell culture” as used herein may beused interchangeably. All of these terms also include their progeny,which are any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.

The term “cell binding element” as used herein is defined as a portionof a protein, which is capable of binding to a receptor on a cellmembrane.

The term “DNA” as used herein is defined as deoxyribonucleic acid.

The term “dendritic cell” or “DC” as used herein is defined as anexample of an antigen presenting cell derived from bone marrow.

The term “epitope” as used herein is defined as small chemical groups onthe antigen molecule that can elicit and react with an antibody. Anantigen can have one or more epitopes. Most antigens have many epitopes;i.e., they are multivalent. In general, an epitope is roughly 5 aminoacids or sugars in size. One skilled in the art understands thatgenerally the overall three-dimensional structure, rather than thespecific linear sequence of the molecule, is the main criterion ofantigenic specificity.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

The term “expression vector” as used herein refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules or ribozymes. Expression vectors can contain avariety of control sequences, which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well and are described infra.

The term “helper T-cell” as used herein is defined as effector T-cellswhose primary function is to promote the activation and functions ofother B and T lymphocytes and of macrophages. Most are CD4 T-cells.

The term “heterologous” as used herein is defined as DNA or RNAsequences or proteins that are derived from the different species.

The term “homologous” as used herein is defined as DNA or RNA sequencesor proteins that are derived from the same species.

The term “host cell” as used herein is defined as cells that areexpressing a heterologous nucleic acid sequence.

The term “immunoglobulin” or “Ig”, as used herein is defined as a classof proteins, which functions as antibodies. The five members included inthis class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA functions asthe primary antibody that is present in body secretions, such as saliva,tears, breast milk, gastrointestinal secretions and mucus secretions ofthe respiratory and genitourinary tracts. IgG functions as the mostcommon circulating antibody. IgM is the main immunoglobulin produced inthe primary response. It is the most efficient immunoglobulin inagglutination, complement fixation, and other antibody responses, and isimportant in defense against bacteria and viruses. IgD is theimmunoglobulin that has no known antibody function, but may serve as anantigen receptor. IgE is the immunoglobulin that mediates immediatehypersensitivity by causing release of mediators from mast cells andbasophils upon exposure to allergen.

The term “major histocompatibility complex”, or “MHC”, as used herein isdefined as a specific cluster of genes, many of which encodeevolutionarily related cell surface proteins involved in antigenpresentation, which are among the most important determinants ofhistocompatibility. Class I MHC, or MHC-I, function mainly in antigenpresentation to CD8 T lymphocytes. Class II MHC, or MHC-II, functionmainly in antigen presentation to CD4 T lymphocytes.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means. Furthermore, one skilled in the art iscognizant that polynucleotides include with limitation mutations of thepolynucleotides, including but not limited to, mutation of thenucleotides, or nucleosides by methods well known in the art.

The term “polypeptide” as used herein is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is mutually inclusive of the terms “peptides” and“proteins”.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

The term “retrogen” or “retrogen fusion protein” as used herein, means apolypeptide having an epitope that is capable of eliciting an immuneresponse in a mammal when expressed and processed as described herein,wherein the polypeptide is fused to a cell binding element.

The term “retrogen expression vector” as used herein refers to theexpression vector comprising at least a polypeptide sequence encoding asignal sequence, an antigen and a cell binding element.

The term “RNA” as used herein is defined as ribonucleic acid.

The term “recombinant DNA” as used herein is defined as DNA produced byjoining pieces of DNA from different sources.

The term “recombinant polypeptide” as used herein is defined as a hybridprotein produced by using recombinant DNA methods.

The term “T-cell” as used herein is defined as a thymus-derived cellthat participates in a variety of cell-mediated immune reactions.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A transformed cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to the polynucleotides to control RNA polymeraseinitiation and expression of the polynucleotides.

The term “vaccine” as used herein is defined as material used to provokean immune response after administration of the materials to a mammal andthus conferring immunity.

The term “virus” as used herein is defined as a particle consisting ofnucleic acid (RNA or DNA) enclosed in a protein coat, with or without anouter lipid envelope, which is only capable of replicating within awhole cell and spreading from cell to cell.

One embodiment of the present invention is an expression vectorcomprising a polynucleotide promoter sequence, a polynucleotide encodinga signal sequence, a polynucleotide encoding an antigen, apolynucleotide encoding a cell binding element, and a polynucleotidepolyadenylation sequence all operatively linked.

In specific embodiments, the nucleic acid sequence encoding a fusionprotein (antigen-cell binding element) is under transcriptional controlof a promoter. Much of the thinking about how promoters are organizedderives from analyses of several viral promoters, including those forthe HSV thymidine kinase (tk) and SV40 early transcription units. Thesestudies, augmented by more recent work, have shown that promoters arecomposed of discrete functional modules, each consisting ofapproximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 genes, a discrete element overlying the start site itselfhelps to fix the place of initiation.

Additional promoter elements, i.e., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the tk promoter, thespacing between promoter elements can be increased to 50 bp apart beforeactivity begins to decline. Depending on the promoter, it appears thatindividual elements can function either co-operatively or independentlyto activate transcription.

A promoter may be one naturally associated with a gene or polynucleotidesequence, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment and/or exon. Such a promoter canbe referred to as “endogenous.” Similarly, an enhancer may be onenaturally associated with a polynucleotide sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding polynucleotidesegment under the control of a recombinant or heterologous promoter,which refers to a promoter that is not normally associated with apolynucleotide sequence in its natural environment. A recombinant orheterologous enhancer refers also to an enhancer not normally associatedwith a polynucleotide sequence in its natural environment. Suchpromoters or enhancers may include promoters or enhancers of othergenes, and promoters or enhancers isolated from any other prokaryotic,viral, or eukaryotic cell, and promoters or enhancers not “naturallyoccurring,” i.e., containing different elements of differenttranscriptional regulatory regions, and/or mutations that alterexpression. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (U.S. Pat. Nos.4,683,202, 5,928,906). Furthermore, it is contemplated the controlsequences that direct transcription and/or expression of sequenceswithin non-nuclear organelles such as mitochondria, chloroplasts, andthe like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. Those of skill inthe art of molecular biology generally know how to use promoters,enhancers, and cell type combinations for protein expression, forexample, see Sambrook et al. (1989). The promoters employed may beconstitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

A promoter sequence exemplified in the experimental examples presentedherein is the immediate early cytomegalovirus (CMV) promoter sequence.This promoter sequence is a strong constitutive promoter sequencecapable of driving high levels of expression of any polynucleotidesequence operatively linked thereto. However, other constitutivepromoter sequences may also be used, including, but not limited to thesimian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV),human immunodeficiency virus (HW) long terminal repeat (LTR) promoter,Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barrvirus immediate early promoter, Rous sarcoma virus promoter, as well ashuman gene promoters such as, but not limited to, the actin promoter,the myosin promoter, the hemoglobin promoter, and the muscle creatinepromoter. Further, the invention should not be limited to the use ofconstitutive promoters. Inducible promoters are also contemplated aspart of the invention. The use of an inducible promoter in the inventionprovides a molecular switch capable of turning on expression of thepolynucleotide sequence which it is operatively linked when suchexpression is desired, or turning off the expression when expression isnot desired. Examples of inducible promoters include, but are notlimited to a metallothionine promoter, a glucocorticoid promoter, aprogesterone promoter, and a tetracycline promoter. Further, theinvention includes the use of a tissue specific promoter, which promoteris active only in a desired tissue. Tissue specific promoters are wellknown in the art and include, but are not limited to, the HER-2 promoterand the PSA associated promoter sequences.

In specific embodiments of the present invention, the expression vectorcomprises a polynucleotide encoding a signal sequence, which directsprocessing of the protein encoded thereby to the appropriate cellularmachinery in order that the protein is secreted from the cell. Exemplarysignal sequences include, but are not limited to, hepatitis B virus Eantigen signal sequence, immunoglobulin heavy chain leader sequences,cytokine leader sequences, and the like, can be used. Essentially, anysignal sequence that directs secretion of a protein from a cell issuitable for use in the expression vector of the invention. In additionto signal sequences, other mechanisms for secretion may be employed,such as but not limited to, truncation or deletion of sequencesinhibiting protein secretion, point mutations of sequences inhibitingprotein secretion, and linkage of the protein to a viral gene to beassembled into viral particles.

An embodiment of the present invention is an expression vector whereinthe polynucleotide encoding an antigen comprises a polynucleotidesequence for at least one epitope, wherein said at least one epitopeinduces a B cell response in a mammal.

A further embodiment of the present invention is an expression vectorwherein the polynucleotide encoding an antigen comprises apolynucleotide sequence for at least one epitope, wherein said at leastone epitope induces a CD4+ T-cell response in a mammal.

Another embodiment of the present invention is an expression vectorwherein the polynucleotide encoding an antigen comprises apolynucleotide sequence for at least one epitope, wherein said at leastone epitope induces a CD8+ T-cell response in a mammal.

A specific embodiment of the present invention is an expression vectorwherein the polynucleotide sequence encoding an antigen comprises apolynucleotide sequence for at least one epitope, wherein said at leastone epitope induces a B cell response, a CD4+ T-cell response and a CD8+T-cell response in a mammal into which said antigen is introduced.

A further specific embodiment of the present invention is an expressionvector wherein the polynucleotide sequence encoding an antigen comprisesa polynucleotide sequence for a plurality of epitopes, wherein saidplurality of epitopes induces a B cell response, a CD4+ T-cell responseand a CD8+ T-cell response in a mammal into which said antigen isintroduced.

In specific embodiments of the present invention, the expression vectorcomprises a polynucleotide sequence encoding an antigen. Thepolynucleotide sequences encoding an antigen are selected from at leastone polynucleotide sequence associated with a disease, wherein saiddisease is selected from the group consisting of infectious disease,cancer and autoimmune disease. More particularly, the polynucleotidesequence encoding the antigen is a polynucleotide sequence selected fromthe group of pathogenic microorganisms that cause infectious diseaseconsisting of virus, bacterium, fungus and protozoan. These DNAsequences encoding known proteins or fragments thereof include viralantigens, such as but not limited to, hepatitis B and hepatitis C virusantigens, human immunodeficiency virus antigens, including but notlimited to, gp160, gp120 and gag proteins, papillomavirus antigens,including but not limited to the E7 and E6 proteins. Herpes virusproteins, such as for example, proteins encoded by Epstein-Barr virus,cytomegalovirus, herpes simplex virus types 1 and 2, and human herpesviruses 6, 7 and 8, are also contemplated in the invention as usefulretrogen fusion proteins. In further embodiments, the polynucleotideencoding an antigen is a polynucleotide selected from the groupconsisting of breast cancer, cervical cancer, melanoma, renal cancer andprostate cancer. In addition, the protein can be one, which inducesactivation of an immune response directed against tumor cells for thepurpose of inhibiting their growth and replication, i.e., tyrosinasethat activates an immune response against melanocytes in melanoma. Othertumor-associated proteins include, but are not limited to, MART, trp,MAGE-1, MAGE-2, MAGE-3, gp100, HER-2, PSA, the Ras antigen associatedwith lung cancer and any other tumor specific, tissue specific or tumorassociated antigens. One skilled in the art is aware of knownpolynucleotide sequences, which encode tumor associated antigens, aswell as, are well documented in the scientific literature and heretoforeunknown polynucleotide sequences are being discovered with greatrapidity. In a further embodiment, the polynucleotide sequence encodingan antigen is selected from an autoimmune disease including, but notlimited to, rheumatoid arthritis, systemic lupus erythematosus, multiplesclerosis, psoriasis and Crohn's disease. In addition, the inventionshould be construed to include DNA that encodes an autoantigen in orderto induce immune tolerance in situations in which such tolerance is ofbenefit to the mammal. Further, the invention should be construed toinclude DNA that encodes an antigen, which is capable of inducing ageneralized immune response in a mammal where a generalized immuneresponse is of benefit to the mammal. A generalized immune response maybe of benefit to the mammal in instances wherein the mammal isimmunosuppressed, i.e., as a result of HIV infection, chemotherapy, orother immunosuppressive procedures. Such antigens may include, but arenot limited to, Fc antibody fragments which when bound to Fc receptorson antigen presenting cells serve to upregulate antigen presentation bythese cells. In addition, interleukins, such as, but not limited to,interleukin 5 may be used to generate a similar adjuvant effect inmammals in which induction of a generalized immune response is desired.The present invention should therefore be construed to include any knownor heretofore unknown polynucleotide sequences which when included inthe expression vector of the invention are capable of activating theimmune response when the vector, or the fusion protein encoded thereby,is introduced into a mammal.

One skilled in the art is cognizant that it is not necessary that thenucleic acid sequence encode a full-length protein. It is simplynecessary that the expressed protein comprise an epitope, which elicitsthe desired immune response when processed in antigen presenting cells.Thus, it is apparent from this information that the nucleic acidsequence may encode any antigen which can elicit an immune response inthe animal into which the expression vector is introduced. Thus, theinvention should in no way be limited to the type of nucleic acidsequences contained within the expression vector, but should include anyand all nucleic acid sequences which are obtained by any means availablein the art, including, without limitation, recombinant means, includingalso without limitation, the cloning of nucleic acid sequences from arecombinant library or a cell genome, using ordinary cloning technologyand PCR™, and the like, and by synthetic means. The invention alsoshould not be construed to be limited in any way to the source of thenucleic acid sequence, in that nucleic acid sequence may be obtainedfrom any available source. One skilled in the art is aware thatprotocols for obtaining a nucleic acid sequence are well known in theart and are described, for example in Sambrook et al. (1989), and inAusubel et al. (1997).

In specific embodiments of the present invention, the expression vectorfurther comprises a polynucleotide sequence encoding a cell bindingelement. The cell binding element is a portion of a polypeptide, whichfacilitates binding of a protein to a cell receptor. A polynucleotideencoding any ligand that binds to a cell receptor protein may be used inthe expression vector of the invention. Exemplary cell binding elementsinclude, but are not limited to, immunoglobulin Fc fragment, toxinreceptor protein cell binding domains, such as for example, thepseudomonas exotoxin cell binding domain, a cytokine, for example,interleukin 5, and interleukin 6, any type of an antibody molecule, andthe like. A skilled artisan is cognizant that any antibody is capable ofbinding to cell surface markers on the surface of antigen presentingcells initiating internalization of the antigen/antibody complex. Thus,an antibody or a fragment thereof can be used as a cell binding elementto initiate internalization. Exemplary antibodies include, but shouldnot be limited to, antiCDC11, antiCD54, antiCD80, and antiCD86.Furthermore, one skilled in the art is cognizant that the cell bindingelement can be homologous or heterologous. For example, the Fc fragmentcan be homologous or heterologous. Thus, the invention should not beconstrued to be limited in any way to the source of the cell bindingelement, in that the sequence for a cell binding element may be obtainedfrom any available source including, without limitation, the cloning ofDNA from a recombinant library or a cell genome, using ordinary cloningtechnology and PCR™, and the like, and by synthetic means.

In addition to using portions of known binding elements, a skilledartisan is cognizant that small peptides could be identified via atypical screening procedure well known in the art. A DNA library (cDNAor genomic) is screened to identify small peptides that bind efficientlyto antigen presenting cells. Once these peptides are identified, thepeptide is sequenced and used as a cell binding element in the presentinvention.

In expression, one will typically include a polyadenylation sequence toeffect proper polyadenylation of the transcript. The nature of thepolyadenylation sequence is not believed to be crucial to the successfulpractice of the invention, and/or any such sequence may be employed.Preferred embodiments include the SV40 polyadenylation sequence, LTRpolyadenylation sequence, and/or the bovine growth hormonepolyadenylation sequence, convenient and/or known to function well invarious target cells. Also contemplated as an element of the expressionvector is a transcriptional termination site. These elements can serveto enhance message levels and/or to minimize read through from theinserted polynucleotide sequences encoding the antigen and cell bindingelements into other sequences of the vector.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast. In instances wherein it is beneficial thatthe expression vector replicate in a cell, the vector may integrate intothe genome of the cell by way of integration sequences, i.e., retroviruslong terminal repeat sequences (LTRs), the adeno-associated virus ITRsequences, which are present in the vector, or alternatively, the vectormay itself comprise an origin of DNA replication and other sequencewhich facilitate replication of the vector in the cell while the vectormaintains an episomal form. For example, the expression vector mayoptionally comprise an Epstein-Barr virus (EBV) origin of DNAreplication and sequences which encode the EBV EBNA-1 protein in orderthat episomal replication of the vector is facilitated in a cell intowhich the vector is introduced. For example, DNA constructs having theEBV origin and the nuclear antigen EBNA-1 coding are capable ofreplication to high copy number in mammalian cells and are commerciallyavailable from, for example, Invitrogen (San Diego, Calif.).

It is important to note that in the present invention it is notnecessary for the expression vector to be integrated into the genome ofthe host cell for proper protein expression. Rather, the expressionvector may also be present in a desired cell in the form of an episomalmolecule. For example, there are certain cell types in which it is notnecessary that the expression vector replicate in order to express thedesired protein. These cells are those which do not normally replicate,such as muscle cells, and yet are fully capable of gene expression. Anexpression vector may be introduced into non-dividing cells and expressthe protein encoded thereby in the absence of replication of theexpression vector.

To identify cells that contain the nucleic acid constructs of thepresent invention, the cells are identified in vitro or in vivo byincluding a marker in the expression vector. Such markers confer anidentifiable change to the cell permitting easy identification of cellscontaining the expression vector. Generally, a selectable marker is onethat confers a property that allows for selection. A positive selectablemarker is one, in which the presence of the marker allows for itsselection, while a negative selectable marker is one in which itspresence prevents its selection. An example of a positive selectablemarker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers in conjunctionwith FACS analysis. For example, NGFR (nerve growth factor receptor) isincluded in the expression vector to facilitate selection of cellscomprising the vector by using a flow cytometric assay detecting NGFRexpression on the cell surface. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

The expression vector may also comprise a prokaryotic origin of DNAreplication and a gene encoding a detectable marker for selection ofprokaryotic cells comprising the expression vector, for example, anantibiotic resistance gene, such as, for example, the ampicillinresistance gene.

In addition, the expression vector may be provided to the cell in theform of RNA instead of DNA. The core components of the vector are thesame as those described herein for a DNA vector, and in addition, othercomponents may be added which serve to stabilize the RNA in bodilyfluids and in tissues and cells.

The actual methods of ligating together the various components describedherein to generate the expression vector of the invention are well knownin the art and are described, for example, in Sambrook et al. (1989),Ausubel et al. (1994), and in Gerhardt et al. (1994).

In specific embodiments, the expression vector is selected from thegroup consisting of viral vectors, bacterial vectors and mammalianvectors. Numerous expression vector systems exist that comprise at leasta part or all of the compositions discussed above. Prokaryote- and/oreukaryote-vector based systems can be employed for use with the presentinvention to produce nucleic acid sequences, or their cognatepolypeptides, proteins and peptides. Many such systems are commerciallyand widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236 and can be bought, for example,under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ BACULOVIRUSEXPRESSION SYSTEM FROM CLONTECH®.

Other examples of expression vector systems include STRATAGENE®'SCOMPLETE CONTROL™ Inducible Mammalian Expression System, which involvesa synthetic ecdysone-inducible receptor, or its pET Expression System,an E. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

A transformed cell comprising an expression vector is generated byintroducing into the cell the expression vector. The introduction of DNAinto a cell or host cell is well known technology in the field ofmolecular biology and is described, for example, in Sambrook et al.(1989), Ausubel et al. (1994), and in Gerhardt et al., (1994). Methodsof transfection of cells include calcium phosphate precipitation,liposome mediated transfection, DEAE dextran mediated transfection,electroporation and the like. Alternatively, cells may be simplytransduced with the retrogen expression vector of the invention usingordinary technology described in the references and examples providedherein. The host cell includes a prokaryotic or eukaryotic cell, and itincludes any transformable organism that is capable of replicating avector and/or expressing a heterologous gene encoded by a vector. A hostcell can, and has been, used as a recipient for vectors. Host cells maybe derived from prokaryotes or eukaryotes, depending upon whether thedesired result is replication of the vector or expression of part or allof the vector-encoded nucleic acid sequences. Numerous cell lines andcultures are available for use as a host cell, and they can be obtainedthrough the American Type Culture Collection (ATCC), which is anorganization that serves as an archive for living cultures and geneticmaterials (www.atcc.org). It is well within the knowledge and skill of askilled artisan to determine an appropriate host. Generally this isbased on the vector backbone and the desired result. A plasmid orcosmid, for example, can be introduced into a prokaryote host cell forreplication of many vectors. Bacterial cells used as host cells forvector replication and/or expression include DH5α, JM109, and KC8, aswell as a number of commercially available bacterial hosts such as SURE®Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla,Calif.). Alternatively, bacterial cells such as E. coli LE392 could beused as host cells for phage viruses. Eukaryotic cells that can be usedas host cells include, but are not limited to yeast, insects andmammals. Examples of mammalian eukaryotic host cells for replicationand/or expression of a vector include, but are not limited to, HeLa,NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Examples of yeast strainsinclude, but are not limited to, YPH499, YPH500 and YPH501. Many hostcells from various cell types and organisms are available and would beknown to one of skill in the art. Similarly, a viral vector may be usedin conjunction with either an eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (1989), and in Ausubel et al.(1994), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,and lentiviruses.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the described host cells to maintain them and to permitreplication of a vector. Also understood and known are techniques andconditions that would allow large-scale production of vectors, as wellas production of the nucleic acids encoded by vectors and their cognatepolypeptides, proteins, or peptides.

A specific embodiment of the present invention is a fusion proteincomprising a signal sequence an antigen and a cell binding element. Theinvention also includes the use of the retrogen protein or fusionprotein as a vaccine. The retrogen protein may be obtained by expressingthe retrogen protein in any cell comprising the expression vector andseparating the retrogen protein from the cell, cell debris and cellmedium. Affinity column purification procedures may be especially usefulfor purification of the retrogen of the invention because the retrogen,by definition comprises a cell binding element. An affinity columncomprising the matching cellular receptor, or a generic protein such asprotein A or protein G, may be used to separate the retrogen from thecellular components. Another embodiment is a vaccine comprising antigenpresenting cells that are transduced in vitro with the fusion protein.

In further embodiments, a vaccine comprises the expression vector,wherein said expression vector comprises a polynucleotide sequenceencoding a promoter sequence, a polynucleotide sequence encoding asecretion signal sequence, a polynucleotide encoding an antigen, apolynucleotide encoding a cell binding element, and a polynucleotideencoding a polyadenylation sequence, all operatively linked. The vaccinecomprising the expression vector is administered directly to the mammalto sites in which there are cells into which the sequences containedwithin the vector may be introduced, expressed and an immune responseagainst the desired protein may be elicited. In this instance, theexpression vector is administered in a pharmaceutical carrier and in aformulation such that the DNA is capable of entering cells, and beingexpressed therein. The expressed protein may then enter antigenpresenting cells for processing and MHC presentation as describedherein. A skilled artisan realizes that the DNA may be given in avariety of ways and, depending upon the route of injection, thecomposition of DNA may need to be manipulated. Exemplary routes ofparenteral injections include, but are not limited to, intramuscular,intraperitoneal, intravenous, subcutaneous and intradermal. Further, itis not necessary that the DNA of the expression vector be introducedinto the cells of the mammal by direct injection of the same into thetissues of the mammal. Rather, other means of introduction of theexpression vector into the mammal may be used, including, but notlimited to non-invasive pressure injection, nasal, oral, etc.

The amount of DNA which is to be introduced into the mammal is an amountsufficient for efficient expression of the DNA in the cell, such that asufficient amount of protein is expressed and secreted therefrom, whichprotein is then taken up by antigen presenting cells and expressedthereon as an MHC complex. Such an amount of DNA is referred to hereinas a “therapeutic amount” of DNA. The precise concentration of DNA whichconstitutes a therapeutic amount may be easily determined by one skilledin the art of administration of such compounds to mammals, and will ofcourse vary depending on the components contained therein, and otherfactors including, but not limited to, the tissue into which the DNA isbeing introduced and the age and health of the mammal.

Another specific embodiment of the present invention is a vaccinecomprising cells that are transduced with the expression vector. Thesetransduced cells are in the form of a pharmaceutical composition foradministration to a mammal for the purpose of eliciting an immuneresponse therein. Expression of the retrogen protein in the cellsresults in secretion of the retrogen protein from the cells. Secretedretrogen protein may then be taken up by antigen presenting cells in themammal for processing therein and expression therefrom as a MHC-I or aMHC-II complex. When the eukaryotic cell is an antigen presenting cell,the retrogen protein may be expressed therein, secreted therefrom andmay reenter the cell for processing and antigenic MHC presentation. Whenthe eukaryotic cell is not an antigen presenting cell, the cellexpresses and secretes the retrogen protein, which is subsequently takenup by an antigen presenting cell for antigenic MHC presentation.Non-antigen presenting cells useful in the invention include any cellwhich does not process antigens for MHC presentation, i.e., musclecells. Antigen presenting cells include dendritic cells (DC),macrophages, monocytes and the like. Tumor cells, which are alsoincluded, may be cells, which are or are not capable of processingantigens for MHC presentation.

The expression vector may also be introduced into stem cells of amammal, either directly in vivo in the mammal, or more preferably, exvivo in cells which are removed from the mammal and are reintroducedinto the mammal following introduction of the vector into the cells. Theexpression vector may also be introduced into other cells in the mammalin an ex vivo approach. When the vector is introduced into cells in themammal, it is not necessary that the vector express the protein encodedthereby immediately, in that, it may be more desirable that the proteinbe expressed in the cells at some later time. In this instance, theexpression vector preferably comprises an inducible promoter, which isactivated upon administration of the appropriate inducer to the mammalor to cells of the mammal. Ex vivo technology is well known in the artand is described, for example, in U.S. Pat. No. 5,399,346.

A further embodiment is an expression vector comprising at least apolynucleotide encoding a signal sequence, a polynucleotide encoding anantigen and a polynucleotide encoding a cell binding domain.

Another embodiment of the present invention is a method to elicit animmune response directed against an antigen. More particularly, thismethod utilizes the expression vector of the present invention tomanipulate cells to produce endogenous antigens as if they wereexogenous antigens. This novel antigen presentation strategy involvestransducing cells with a novel recombinant expression vector to produceand secrete a fusion-protein consisting of an antigen and a cell-bindingelement. The secreted fusion protein is endocytosed or “retrogradely”transported into antigen presenting cells via receptor-mediatedendocytosis (Daeron, 1997; Serre et al., 1998; Ravetch et al., 1993). Asa result, the fusion protein, or “retrogen” as termed in the presentdisclosure because of its retrograde transport following secretion, isprocessed in the endosomal pathway and is presented on the cell surfaceof the antigen presenting cells as an MHC-II restricted exogenousantigenic fragments even though it has been produced endogenously. TheMHC-II bound antigenic fragments of the antigen on the surface of theantigen presenting cells activate CD4+ T-cells that in turn stimulateCD8+ T-cells and macrophages, as well as B-cells to induce both cellularand humoral immunity.

It has also been discovered in the present invention that the retrogenprotein may also be processed in the cytosolic pathway during the fusionprotein synthesis, secretion and endocytosis and become associated withMHC-I on the surface of the antigen presenting cells to directlyactivate CD8+ T-cells. Activation of CD8+ T cells by internalizedantigens is described in the art and for example, inKovacsovics-Bankowski et al., 1995. In addition, as noted above anddescribed in more detail elsewhere herein, B cells may be activated bythe secreted retrogen. Thus, B cell activation is enhanced markedly inthe present system in that CD4+ cells also activates B cells. Thus, thisstrategy uses a unifying mechanism to activate all of the arms of theimmune system.

In specific embodiments, the expression vector is introduced into a cellto produce a transduced cell. Expression of the retrogen protein in thecells results in secretion of the retrogen protein from the cells.Secreted retrogen protein may then be taken up by antigen presentingcells in the mammal for processing therein and expression therefrom as aMHC-I or a MHC-II complex. Thus, one skilled in the art realizes thatthe transduced cell or first cell, secretes the antigen and the secretedantigen is internalized into a cell, a second cell, either the same cellor a different cell. When the eukaryotic cell is an antigen presentingcell, the retrogen protein may be expressed therein, secreted therefromand may reenter the cell for processing and antigenic MHC presentation.When the eukaryotic cell is not an antigen presenting cell, the cellexpresses and secretes the retrogen protein, which is subsequently takenup by an antigen presenting cell for antigenic MHC presentation.Non-antigen presenting cells useful in the invention include any cellwhich does not process antigens for MHC presentation, i.e., musclecells. Antigen presenting cells include dendritic cells (DC),macrophages, monocytes and the like. Tumor cells, which are alsoincluded, may be cells, which are or are not capable of processingantigens for MHC presentation.

A further embodiment of the present invention, is a method to elicit animmune response directed against an antigen comprising the step ofadministering the expression vector directly to a mammal.

The invention also includes a method of screening or identifying apolynucleotide sequence which encodes at least one MHC-II restrictedepitope that is capable of eliciting an immune response in a mammal.Preferably, the polypeptide, which is identified, is one which elicitsan immune response that is beneficial to the mammal. The methodcomprises obtaining a population of isolated DNA molecules and screeningfor those isolated DNA molecules which encode at least one MHC-IIrestricted epitope that is capable of activating CD4+ helper T-cells.The DNA molecules are referred to herein as “test DNA” or “testpolynucleotide sequence.” The test polynucleotide sequences are clonedinto the expression vector of the present invention, in the vector whichis positioned between the signal sequence and the cellular bindingelement as depicted, for example, in FIG. 25. In the method, antigenpresenting cells are transduced by introducing the vector comprising thetest polynucleotide sequence into the antigen presenting cells,transduced antigen presenting cells are contacted with naive T-cells orprimed T-cells and the ability of the transduced cells to activate naiveCD4+ T cells in vitro is assessed by assessing whether any naive T-cellsor primed T-cells are activated upon contact with said transducedantigen presenting cell. Activation of T cells by transduced antigenpresenting cells is an indication that the test polynucleotide sequencecontained therein is a polynucleotide sequence, or gene or fragmentthereof which encodes at least one epitope capable of activating CD4+helper T-cells to elicit an immune response in a mammal. Suitablecontrols which can be used in the assay include cells which aretransduced with an expression vector comprising an isolatedpolynucleotide sequence which is known not to activate the immuneresponse in an mammal (negative control), and cells which are transducedwith an expression vector comprising an isolated polynucleotide sequencewhich is known to activate the immune response in an mammal (positivecontrol). One skilled in the art is cognizant that this screeningprocedure can be utilized to screen the human genome to identify genesthat encode proteins or epitopes that are recognized by CD4+ T-cellsthat could be used for immunotherapy for cancer or autoimmune disease orfor gene therapy. Furthermore, other genomes can be screened includingbacterial, viral, or parasitic.

The in vitro T-cell activation assay may be adapted to be ahigh-throughput automated assay in order to facilitate the testing ofmany different test polynucleotide sequences at one time. One skilled inthe art recognizes that the present invention can be manipulated totransduce cells with expression vectors containing a variety of possibleepitope sequences. The transduced cells may be placed in 96-well plates,containing naive T-cells, and the activation of the T-cells may beassessed by automated assessment of incorporation of radioactivity intothe DNA of the T-cells, using technology readily available in clinicalimmunology.

In further embodiments, the protein product encoded by the testpolynucleotide sequences may be further evaluated to assess activationof the immune response in a mammal in vivo. This assay is the same asthe in vitro assay except, the transduced antigen presenting cells thatwere transduced by introducing the expression vector comprising the testpolynucleotide sequences are administered to a mammal via a parenteralroute. In specific embodiments, the expression vector comprising thetest polynucleotide sequences is administered directly to a mammal.T-cells are collected from splenocytes and co-cultured with dendriticcells. The activation of T-cells is assessed to determine if the testpolynucleotide encoding the test polypeptide is a capable of activatingCD4+ helper T cells. Furthermore, one skilled in the art is cognizantthat this screening procedure could be utilized to identify MHC-IIrestricted epitopes that could be use to treat cancer, viral infectionsand autoimmune disease.

As noted herein, the test polynucleotide sequences may be obtained byany ordinary means common in the art of molecular biology. For example,test polynucleotide sequences may be obtained from an expressionlibrary, which library may express proteins whose function is unknown.Test polynucleotide sequences may also be obtained from an expressionlibrary which expresses proteins of known function, but which have notheretofore been known to possess the property of activation of theimmune system in an mammal. Exemplary expression cDNA libraries include,but are not limited to, tumor cells, viral genomes, bacterial genomes,parasitic genomes, and human genomes. Test polynucleotide sequences mayalso be obtained using combinatorial methodology, wherein it is notknown at the outset whether the polynucleotide sequence encodes aprotein, and moreover, it is not known whether the polynucleotidesequence encodes a protein which is capable of activating the immuneresponse. Test polynucleotide sequences may also be obtained bysynthetic methods, wherein a polynucleotide sequence is synthesized inan automated synthesizer, fragments of discrete lengths are cloned intothe expression vector and are tested as described herein.

It is not always necessary that the immune response be protective, butmerely that it be beneficial to the host mammal. For example, it may bebeneficial to a mammal to induce immune tolerance in situations whereinan immune response to an antigen is detrimental to the mammal, forexample, in certain autoimmune diseases such as rheumatoid arthritis,systemic lupus erythrematosus, psoriasis, multiple sclerosis, Crohn'sdisease, etc., a diminution in the immune response is desired which canbe achieved by inducing immune tolerance against the offensive antigen.In this instance, the DNA comprises DNA encoding the offensive antigenwhich is then expressed in cells of the mammal and subsequentlyprocessed in antigen presenting cells so as to be expressed on thesurface thereof as an MHC-I and/or an MHC-II complex in order to induceimmune tolerance in the mammal against the antigen.

In a further embodiment, an identified polynucleotide sequence is usedas a method of treating cancer, viral infection or an autoimmunedisease. More particularly, the identified polynucleotide encoding atest polypeptide is transduced into antigen presenting cells and thetransduced antigen presenting cells are administered directly to amammal via a parenteral route to treat cancer, a viral infection or anautoimmune disease. Furthermore, the expression vector containing atleast the polynucleotide encoding a test polypeptide and a cell bindingelement is administered directly into a mammal via a parenteral route totreat cancer, a viral infection or an autoimmune disease.

A further embodiment of the present invention is a method of producing avaccine to immunize a mammal comprising the steps of: transducingantigen presenting cell by introducing the expression vector of thepresent invention into a cell and expressing said vector to produce anantigen under conditions wherein said antigen is secreted from the cell.In specific embodiments, antigen presenting cells are transduced withthe antigen in vitro or ex vivo prior to administering the antigenpresenting cells to the mammal. All of the vaccines of the presentinvention can be administered parenterally.

In specific embodiments, the method of inducing an immune responsecomprises the step of co-administering to an organism the expressionvector and a cytokine expression vector. A number of studies have shownthat the responses to individual plasmids can be enhanced byco-administration of a cytokine expressing plasmid. It should be notedthat picogram to nanogram quantities of locally synthesized cytokinefrom the expression vector are too low to have systemic effects on thewhole mammal, but can still strongly influence the local cytokineenvironment and thus the immune response to the administered antigen.Examples of cytokines include, but are not limited to, GM-CSF and IL-2.A skilled artisan readily recognizes that the polynucleotide sequencesfor a cytokine and the polynucleotide sequences for the antigen can beincorporated into one expression vector; thus eliminating the use of twoseparate vectors. In addition to cytokines, plasmids that containunmethylated CpG sequences enhance the cell mediated (Th1) response(Carson et al., 1997). CpG sequence motifs include but are not limitedto, RRCpGYY. Thus, a skilled artisan realizes that supplementation of acytokine with the expression vector or addition of a CpG sequence motifin the present invention would result in the enhancement of the immuneresponse.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together; each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple nucleic acid sequences can beefficiently expressed using a single promoter/enhancer to transcribe asingle message (U.S. Pat. Nos. 5,925,565 and 5,935,819). Furthermore, askilled artisan is cognizant that the entire nucleic acid sequence of agene does not have to be used. Instead, partial nucleic acid sequencesof MHC class I and II restricted epitopes can be fused together,resulting in a chimeric fusion gene transcribed by one promoter. Forexample, a specific embodiment of the present invention is a method ofsimultaneously inducing both CD4+ and CD8+ T-cells comprising the stepsof administering a fusion protein wherein the protein comprises both aMHC-I and MHC-II epitope fused to a cell binding element. Thus, oneskilled in the art recognizes that the use of multiple antigenicsequences results in the treatment of a variety of diseases with theadministration of one vaccine.

Another specific embodiment of the present invention is a method ofinducing an immune response comprising the steps of administering to amammal one expression vector, wherein said expression vector comprises apolynucleotide sequence encoding a first fusion protein and apolynucleotide sequence encoding a second fusion protein undertranscriptional control of one promoter, wherein said first fusionprotein comprises a first signal sequence, a first antigen and a firstcell binding element and said second fusion protein comprises a secondsignal sequence, a second antigen and a first cell binding element. Inspecific embodiments, the first and second signal sequences are the samesignal sequence, the first and second antigens are different antigensand the cell binding elements is a Fc fragment. In further embodiments,the first and second signal sequences are the same, the first and secondantigens are different antigens and the first and second cell bindingelements are the same cell binding elements. Further embodimentsinclude, the first and second signal sequences are different, the firstand second antigens are different antigens and the first and second cellbinding elements are the same cell binding elements or the first andsecond signal sequences are the same, the first and second antigens aredifferent antigens and the first and second cell binding elements aredifferent cell binding elements. An additional embodiment includes thatthe polynucleotide sequence encoding the first fusion protein and thepolynucleotide sequence encoding the second fusion protein are underseparate-transcriptional control, and wherein the polynucleotidesequence encoding the first fusion protein and the polynucleotidesequence encoding the second fusion protein are in tandem in oneexpression vector.

One skilled in the art is cognizant that multiple nucleic acid sequencescan be cloned into the vector in tandem such that each nucleic acidsequence is a separate entity. Each entity contains a promoter thatdrives the expression of the individual nucleic acid sequence resultingin expression of separate antigens from one vector. This techniqueefficiently expresses nucleic acid sequences using multiple promoters totranscribe the individual messages.

A further embodiment of the present invention is a method of producing afusion protein comprising the steps of introducing the expression vectorof the present invention into a cell and expressing said vector toproduce a fusion protein under conditions wherein said fusion protein issecreted from the cell. In specific embodiments, antigen presentingcells are transduced with the fusion protein in vitro. Moreparticularly, the fusion protein is administered parenterally to amammal.

A specific embodiment of the present invention is a method of secretingan intracellular protein comprising the steps of introducing anexpression vector into a cell, wherein said expression vector comprisesa polynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding an intracellular protein, apolynucleotide encoding a cell binding element, and a polynucleotidepolyadenylation sequence, all operatively linked and expressing saidvector to produce a fusion protein under conditions wherein said fusionprotein is secreted from the cell. More specifically, the polynucleotidesequence encoding the intracellular protein is truncated or mutated toincrease efficiency of secretion. In specific embodiments, theintracellular protein is HPV 16 E7.

Another specific embodiment of the present invention is a method ofsecreting a membrane protein comprising the steps of introducing anexpression vector into a cell, wherein said expression vector comprisesa polynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding a membrane protein, a polynucleotideencoding a cell binding element, and a polynucleotide polyadenylationsequence, all operatively linked and expressing said vector to produce afusion protein under conditions wherein said fusion protein is secretedfrom the cell. More specifically, the polynucleotide sequence encodingthe membrane protein is truncated or mutated to increase efficiency ofsecretion. In specific embodiments, the membrane protein is EBV nuclearantigen 1.

The invention also includes a kit comprising the composition of theinvention and an instructional material that describes adventitiallyadministering the composition to a cell or a tissue of a mammal. Inanother embodiment, this kit comprises a (preferably sterile) solventsuitable for dissolving or suspending the composition of the inventionprior to administering the compound to the mammal.

Dosage and Formulation

The expression vectors, transduced cells and fusion proteins (activeingredients) of this invention can be formulated and administered totreat a variety of disease states by any means that produces contact ofthe active ingredient with the agent's site of action in the body of theorganism. They can be administered by any conventional means availablefor use in conjunction with pharmaceuticals, either as individualtherapeutic active ingredients or in a combination of therapeutic activeingredients. They can be administered alone, but are generallyadministered with a pharmaceutical carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

The active ingredient can be administered orally in solid dosage formssuch as capsules, tablets and powders, or in liquid dosage forms such aselixirs, syrups, emulsions and suspensions. The active ingredient canalso be formulated for administration parenterally by injection, rapidinfusion, nasopharyngeal absorption or dermoabsorption. The agent may beadministered intramuscularly, intravenously, or as a suppository.

Gelatin capsules contain the active ingredient and powdered carrierssuch as lactose, sucrose, mannitol, starch, cellulose derivatives,magnesium stearate, stearic acid, and the like. Similar diluents can beused to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, suitable oil, saline, aqueous dextrose (glucose), andrelated sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration contain the active ingredient,suitable stabilizing agents and, if necessary, buffer substances.Antioxidizing agents such as sodium bisulfate, sodium sulfite orascorbic acid, either alone or combined, are suitable stabilizingagents. Also used are citric acid and its salts and sodiumEthylenediaminetetraacetic acid (EDTA). In addition, parenteralsolutions can contain preservatives such as benzalkonium chloride,methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences, astandard reference text in this field.

The active ingredients of the invention may be formulated to besuspended in a pharmaceutically acceptable composition suitable for usein mammals and in particular, in humans. Such formulations include theuse of adjuvants such as muramyl dipeptide derivatives (MDP) or analogsthat are described in U.S. Pat. Nos. 4,082,735; 4,082,736; 4,101,536;4,185,089; 4,235,771; and 4,406,890. Other adjuvants, which are useful,include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate anddimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, and IL-12.Other components may include a polyoxypropylene-polyoxyethylene blockpolymer (Pluronic®), a non-ionic surfactant, and a metabolizable oilsuch as squalene (U.S. Pat. No. 4,606,918).

Additionally, standard pharmaceutical methods can be employed to controlthe duration of action. These are well known in the art and includecontrol release preparations and can include appropriate macromolecules,for example polymers, polyesters, polyamino acids, polyvinyl,pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethylcellulose or protamine sulfate. The concentration of macromolecules aswell as the methods of incorporation can be adjusted in order to controlrelease. Additionally, the agent can be incorporated into particles ofpolymeric materials such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylenevinylacetate copolymers. In addition to beingincorporated, these agents can also be used to trap the compound inmicrocapsules.

Useful pharmaceutical dosage forms for administration of the compoundsof this invention can be illustrated as follows.

Capsules: Capsules are prepared by filling standard two-piece hardgelatin capsulates each with 100 milligram of powdered activeingredient, 175 milligrams of lactose, 24 milligrams of talc and 6milligrams magnesium stearate.

Soft Gelatin Capsules: A mixture of active ingredient in soybean oil isprepared and injected by means of a positive displacement pump intogelatin to form soft gelatin capsules containing 100 milligrams of theactive ingredient. The capsules are then washed and dried.

Tablets: Tablets are prepared by conventional procedures so that thedosage unit is 100 milligrams of active ingredient. 0.2 milligrams ofcolloidal silicon dioxide, 5 milligrams of magnesium stearate, 275milligrams of microcrystalline cellulose, 11 milligrams of cornstarchand 98.8 milligrams of lactose. Appropriate coatings may be applied toincrease palatability or to delay absorption.

Injectable: A parenteral composition suitable for administration byinjection is prepared by stirring 1.5% by weight of active ingredientsin 10% by volume propylene glycol and water. The solution is madeisotonic with sodium chloride and sterilized.

Suspension: An aqueous suspension is prepared for oral administration sothat each 5 milliliters contain 100 milligrams of finely divided activeingredient, 200 milligrams of sodium carboxymethyl cellulose, 5milligrams of sodium benzoate, 1.0 grams of sorbitol solution U.S.P. and0.025 milliliters of vanillin.

Accordingly, the pharmaceutical composition of the present invention maybe delivered via various routes and to various sites in an mammal bodyto achieve a particular effect (see, e.g., Rosenfeld et al., 1991;Rosenfeld et al., 1991 a; Jaffe et al., supra; Berkner, supra). Oneskilled in the art will recognize that although more than one route canbe used for administration, a particular route can provide a moreimmediate and more effective reaction than another route. Local orsystemic delivery can be accomplished by administration comprisingapplication or instillation of the formulation into body cavities,inhalation or insufflation of an aerosol, or by parenteral introduction,comprising intramuscular, intravenous, peritoneal, subcutaneous,intradermal, as well as topical administration.

The active ingredients of the present invention can be provided in unitdosage form wherein each dosage unit, e.g., a teaspoonful, tablet,solution, or suppository, contains a predetermined amount of thecomposition, alone or in appropriate combination with other activeagents. The term “unit dosage form” as used herein refers to physicallydiscrete units suitable as unitary dosages for human and mammalsubjects, each unit containing a predetermined quantity of thecompositions of the present invention, alone or in combination withother active agents, calculated in an amount sufficient to produce thedesired effect, in association with a pharmaceutically acceptablediluent, carrier, or vehicle, where appropriate. The specifications forthe unit dosage forms of the present invention depend on the particulareffect to be achieved and the particular pharmacodynamics associatedwith the pharmaceutical composition in the particular host.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

Lipid Formulation and/or Nanocapsules

In certain embodiments, the use of lipid formulations and/ornanocapsules is contemplated for the introduction of the expressionvector, into host cells.

Nanocapsules can generally entrap compounds in a stable and/orreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and/or such particles maybe easily made.

In a specific embodiment of the invention, the expression vector may beassociated with a lipid. The expression vector associated with a lipidmay be encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Thelipid or lipid/expression vector associated compositions of the presentinvention are not limited to any particular structure in solution. Forexample, they may be present in a bilayer structure, as micelles, orwith a “collapsed” structure. They may also simply be interspersed in asolution, possibly forming aggregates which are not uniform in eithersize or shape.

Lipids are fatty substances which may be naturally occurring orsynthetic lipids. For example, lipids include the fatty droplets thatnaturally occur in the cytoplasm as well as the class of compounds whichare well known to those of skill in the art which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Phospholipids may be used for preparing the liposomes according to thepresent invention and may carry a net positive, negative, or neutralcharge. Diacetyl phosphate can be employed to confer a negative chargeon the liposomes, and stearylamine can be used to confer a positivecharge on the liposomes. The liposomes can be made of one or morephospholipids.

A neutrally charged lipid can comprise a lipid with no charge, asubstantially uncharged lipid, or a lipid mixture with equal number ofpositive and negative charges. Suitable phospholipids includephosphatidyl cholines and others that are well known to those of skillin the art.

Lipids suitable for use according to the present invention can beobtained from commercial sources. For example, dimyristylphosphatidylcholine (“DMPC”) can be obtained from Sigma Chemical Co.,dicetyl phosphate (“DCP”) is obtained from K & K Laboratories(Plainview, N.Y.); cholesterol (“Chol”) is obtained fromCalbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and otherlipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Preferably, chloroform is used as theonly solvent since it is more readily evaporated than methanol.

Phospholipids from natural sources, such as egg or soybeanphosphatidylcholine, brain phosphatidic acid, brain or plantphosphatidylinositol, heart cardiolipin and plant or bacterialphosphatidylethanolamine are preferably not used as the primaryphosphatide, i.e., constituting 50% or more of the total phosphatidecomposition, because of the instability and leakiness of the resultingliposomes.

“Liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes may be characterized as havingvesicular structures with a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). However, the present invention also encompassescompositions that have different structures in solution than the normalvesicular structure. For example, the lipids may assume a micellarstructure or merely exist as nonuniform aggregates of lipid molecules.Also contemplated are lipofectamine-nucleic acid complexes.

Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios the liposome is the preferred structure. The physicalcharacteristics of liposomes depend on pH, ionic strength and/or thepresence of divalent cations. Liposomes can show low permeability toionic and/or polar substances, but at elevated temperatures undergo aphase transition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and/or results in an increase inpermeability to ions, sugars and/or drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and/or neutrophils; adsorption to the cell surface, eitherby nonspecific weak hydrophobic and/or electrostatic forces, and/or byspecific interactions with cell-surface components; fusion with theplasma cell membrane by insertion of the lipid bilayer of the liposomeinto the plasma membrane, with simultaneous release of liposomalcontents into the cytoplasm; and/or by transfer of liposomal lipids tocellular and/or subcellular membranes, and/or vice versa, without anyassociation of the liposome contents. Varying the liposome formulationcan alter which mechanism is operative, although more than one mayoperate at the same time.

Liposome-mediated oligonucleotide delivery and expression of foreign DNAin vitro has been very successful. Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987)accomplished successful liposome-mediated gene transfer in rats afterintravenous injection.

In certain embodiments of the invention, the lipid may be associatedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the lipid may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the lipid may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression vectorshave been successfully employed in transfer and expression of anoligonucleotide in vitro and in vivo, then they are applicable for thepresent invention. Where a bacterial promoter is employed in the DNAconstruct, it also will be desirable to include within the liposome anappropriate bacterial polymerase.

Liposomes used according to the present invention can be made bydifferent methods. The size of the liposomes varies depending on themethod of synthesis. A liposome suspended in an aqueous solution isgenerally in the shape of a spherical vesicle, having one or moreconcentric layers of lipid bilayer molecules. Each layer consists of aparallel array of molecules represented by the formula XY, wherein X isa hydrophilic moiety and Y is a hydrophobic moiety. In aqueoussuspension, the concentric layers are arranged such that the hydrophilicmoieties tend to remain in contact with an aqueous phase and thehydrophobic regions tend to self-associate. For example, when aqueousphases are present both within and without the liposome, the lipidmolecules may form a bilayer, known as a lamella, of the arrangementXY-YX. Aggregates of lipids may form when the hydrophilic andhydrophobic parts of more than one lipid molecule become associated witheach other. The size and shape of these aggregates will depend upon manydifferent variables, such as the nature of the solvent and the presenceof other compounds in the solution.

Liposomes within the scope of the present invention can be prepared inaccordance with known laboratory techniques. In one preferredembodiment, liposomes are prepared by mixing liposomal lipids, in asolvent in a container, e.g., a glass, pear-shaped flask. The containershould have a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

Dried lipids can be hydrated at approximately 25-50 mM phospholipid insterile, pyrogen-free water by shaking until all the lipid film isresuspended. The aqueous liposomes can be then separated into aliquots,each placed in a vial, lyophilized and sealed under vacuum.

In the alternative, liposomes can be prepared in accordance with otherknown laboratory procedures: the method of Bangham et al. (1965), thecontents of which are incorporated herein by reference; the method ofGregoriadis, as described in DRUG CARRIERS IN BIOLOGY AND MEDICINE, G.Gregoriadis ed. (1979) pp. 287-341, the contents of which areincorporated herein by reference; the method of Deamer and Uster, 1983,the contents of which are incorporated by reference; and thereverse-phase evaporation method as described by Szoka andPapahadjopoulos, 1978. The aforementioned methods differ in theirrespective abilities to entrap aqueous material and their respectiveaqueous space-to-lipid ratios.

The dried lipids or lyophilized liposomes prepared as described abovemay be dehydrated and reconstituted in a solution of inhibitory peptideand diluted to an appropriate concentration with an suitable solvent,e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer.Unencapsulated nucleic acid is removed by centrifugation at 29,000×g andthe liposomal pellets washed. The washed liposomes are resuspended at anappropriate total phospholipid concentration, e.g., about 50-200 mM. Theamount of nucleic acid encapsulated can be determined in accordance withstandard methods. After determination of the amount of nucleic acidencapsulated in the liposome preparation, the liposomes may be dilutedto appropriate concentrations and stored at 4° C. until use.

A pharmaceutical composition comprising the liposomes will usuallyinclude a sterile, pharmaceutically acceptable carrier or diluent, suchas water or saline solution.

Gene Therapy Administration

One skilled in the art recognizes that the expression vector of thepresent invention can be utilized for gene therapy. For gene therapy, askilled artisan would be cognizant that the vector to be utilized mustcontain the gene of interest operatively linked to a promoter. Forantisense gene therapy, the antisense sequence of the gene of interestwould be operatively linked to a promoter. One skilled in the artrecognizes that in certain instances other sequences such as a 3′ UTRregulatory sequences are useful in expressing the gene of interest.Where appropriate, the gene therapy vectors can be formulated intopreparations in solid, semisolid, liquid or gaseous forms in the waysknown in the art for their respective route of administration. Meansknown in the art can be utilized to prevent release and absorption ofthe composition until it reaches the target organ or to ensuretimed-release of the composition. A pharmaceutically acceptable formshould be employed which does not ineffectuate the compositions of thepresent invention. In pharmaceutical dosage forms, the compositions canbe used alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. A sufficient amount ofvector containing the therapeutic nucleic acid sequence must beadministered to provide a pharmacologically effective dose of the geneproduct.

One skilled in the art recognizes that different methods of delivery maybe utilized to administer a vector into a cell. Examples include: (1)methods utilizing physical means, such as electroporation (electricity),a gene gun (physical force) or applying large volumes of a liquid(pressure); and (2) methods wherein said vector is complexed to anotherentity, such as a liposome, aggregated protein or transporter molecule.

Accordingly, the present invention provides a method of transferring atherapeutic gene to a host, which comprises administering the vector ofthe present invention, preferably as part of a composition, using any ofthe aforementioned routes of administration or alternative routes knownto those skilled in the art and appropriate for a particularapplication. Effective gene transfer of a vector to a host cell inaccordance with the present invention to a host cell can be monitored interms of a therapeutic effect (e.g. alleviation of some symptomassociated with the particular disease being treated) or, further, byevidence of the transferred gene or expression of the gene within thehost (e.g., using the polymerase chain reaction in conjunction withsequencing, Northern or Southern hybridizations, or transcription assaysto detect the nucleic acid in host cells, or using immunoblot analysis,antibody-mediated detection, mRNA or protein half-life studies, orparticularized assays to detect protein or polypeptide encoded by thetransferred nucleic acid, or impacted in level or function due to suchtransfer).

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

Furthermore, the actual dose and schedule can vary depending on whetherthe compositions are administered in combination with otherpharmaceutical compositions, or depending on interindividual differencesin pharmacokinetics, drug disposition, and metabolism. Similarly,amounts can vary in in vitro applications depending on the particularcell line utilized (e.g., based on the number of vector receptorspresent on the cell surface, or the ability of the particular vectoremployed for gene transfer to replicate in that cell line). Furthermore,the amount of vector to be added per cell will likely vary with thelength and stability of the therapeutic gene inserted in the vector, aswell as also the nature of the sequence, and is particularly a parameterwhich needs to be determined empirically, and can be altered due tofactors not inherent to the methods of the present invention (forinstance, the cost associated with synthesis). One skilled in the artcan easily make any necessary adjustments in accordance with theexigencies of the particular situation.

It is possible that cells containing the therapeutic gene may alsocontain a suicide gene (i.e., a gene which encodes a product that can beused to destroy the cell, such as herpes simplex virus thymidinekinase). In many gene therapy situations, it is desirable to be able toexpress a gene for therapeutic purposes in a host cell but also to havethe capacity to destroy the host cell once the therapy is completed,becomes uncontrollable, or does not lead to a predictable or desirableresult. Thus, expression of the therapeutic gene in a host cell can bedriven by a promoter although the product of said suicide gene remainsharmless in the absence of a prodrug. Once the therapy is complete or nolonger desired or needed, administration of a prodrug causes the suicidegene product to become lethal to the cell. Examples of suicidegene/prodrug combinations which may be used are Herpes SimplexVirus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU;oxidoreductase and cycloheximide; cytosine deaminase and5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) andAZT; and deoxycytidine kinase and cytosine arabinoside.

The following examples are offered by way of example, and are notintended to limit the scope of the invention in any manner.

EXAMPLE 1 Construction and Expression of HBe Antigen in a RetroviralVector

Although both HBcAg and HBeAg proteins are encoded by the HBV pre-CCgene, the secretory HBeAg protein is initiated at a start codon twentynine residues upstream of the start codon for HBcAg. The HBeAg geneobtained from the American Type Culture Collection (Rockville, Md.) wasrepaired to correct two mutations. The two mutations were found to occurfrom a single base pair deletion, which caused a frameshift at codon 74,resulting in two consecutive stop codons at 84 and 85. These mutationswere corrected by inserting the deleted base using PCR mutagenesis. Thearginine-rich, C′-terminal sequence of HBeAg (aa 150-185) which iscleaved during viral infection was deleted. The truncated HBeAg gene wasthen fused in-frame with an IgG Fc fragment. The HBe-retrogen fusiongene (HBe-retrogen) was cloned into the retroviral vector (LNC-NGFR), orthe expression vector pRc/CMV. The vectors comprising HBcAg (cytosolic)and HBeAg (secretory) were constructed using technology available in theart and described, for example in, Sambrook et al. (1989) and in Ausubelet al. (1997). The IgG Fc fragment gene was fused with an IgG signalleader sequence and was cloned into the expression vectors as shown inFIG. 2A. In this manner, a series of control retroviral vectorscontaining the HBeAg gene (secretory), the Fc fragment gene with asignal leader sequence (secretory), or the HBcAg gene (cytosolic), wereconstructed as represented in FIG. 2A.

To assess the expression and secretion of the HBe-Fc fusion protein, COScells were transfected with various expression vectors and 48 hourslater, the cells were radiolabeled. As shown in FIG. 3, a protein bandcorresponding to the HBeAg-Fc fusion protein, was detected in both celllysates and culture medium when either was precipitated with ananti-human IgG or anti-HBeAg antibody (Sigma Chemical Co. St. Louis,Mo.).

Immunofluorescent staining of transfected cells also exhibited a typicalsecretory protein pattern. The HBcAg proteins were only observed in thecell lysates obtained from the transfected cells, and the HBeAg and Fcfragment proteins were observed in both the culture medium and the celllysates. These results indicate that the HBeAg-Fc proteins(HBe-retrogen) are efficiently produced and secreted from cells.

EXAMPLE 2 Transduction and Expression of HBe-Retrogen in Dentritic Cells(DCs)

To assess the “retrogen” strategy in DCs, the retroviral vectorscontaining the HBe-retrogen or various control genes including a NGFRmarker (FIG. 2A) were produced from PA317 packaging cells using thetransient transfection. Murine bone marrow cells were obtained fromC57BL/B6 mice. The cells were stimulated in culture medium supplementedwith murine stem cell factor (SCF) and IL-6, and were transduced withthe retroviral vectors as described infra, et al.

Transduction of DCs

PA317 packaging cells obtained from the American Type Culture Collection(Rockville, Md.) at approximately 40% confluency were cultured in 100 mmculture dishes with Dulbecco's Modified Eagle Media (DMEM) containing10% fetal bovine serum (FBS). The cells were transfected with 10-15 μmfilter.

Murine bone marrow was flushed from the bones of the mouse limbs, passedthrough a nylon mesh and depleted of red cells with ammonium chloride.After extensive washing with RPMI-1640, the cells were incubated withrabbit complement and a set of cocktail of monoclonal antibodies,anti-CD4+, anti-CD8+, anti-B cells and anti-MHC class positive cells inRPMI-1640 at 37° C. for 40-60 minutes. After extensive washing withRPMI-1640, 5×10⁵ cells/ml were suspended in RPMI-1640 supplemented with6% FBS, 80 ng mSCF/ml and 20 U rmIL-6/ml. The cells were plated in12-well culture plates (3 ml/well), incubated at 37° C. and 5% CO₂overnight, and then replaced with the fresh medium comprising the sameingredients. After 48-hour incubation, the cells were collected bycentrifugation, resuspended in 1.5 ml of the retrovirus supernatants,and placed onto 24-well culture plates, which were coated withRetronectin at a concentration of 10 mg/ml. The cells were centrifugedat 2,500 rpm at 37° C. for 90-120 minutes, and were then incubated at37° C. and 5% CO₂ for an additional 3-4 hours for retroviraltransduction. The retrovirus supernatants were then replaced with 2.5 mlof RPMI-1640 supplemented with 5% FBS, 10 ng mSCF/ml, 60 ng mGM-CSF/mland 100 U mIL-4/ml (R&D Systems, Minneapolis, Minn.) overnight. Thetransduction procedure was then repeated 2-3 times. After transduction,the cells were washed and cultured in Opti-MEM (GIBCO, Grand Island,N.Y.) containing mGM-CSF and mIL-4 for several days in order to furthermature the DCs prior to harvest (Banchereau et al., 1998; Inaba et al.,1992).

Evaluation of Transduction (Measurement of Expression)

After several days in culture, the cells exhibited typical DC morphologyand high levels of MHC, adhesion, and co-stimulation molecules (CD11,CD54, CD80 and CD86) were expressed on the bone-marrow-derived DCs(FIGS. 4A-4C and 5A-5E). About 20 to 30% of the cells in the culturewere transduced, as determined by anti-NGFR staining. Transcription ofthe HBe-retrogen gene in the DCs was demonstrated in a RT-PCR assay.

The RT-PCR assay was performed as follows: Cellular RNA was extractedfrom the DCs using Trizoal (Gibco-BRL Grand Island, N.Y.) and wastreated with RNASE-free DNASE at 37° C. for 30 minutes. After reversetranscription, the cDNAs were used as templates for a PCR reaction usinga pair of primers corresponding to the HBeAg gene. The PCR products wereanalyzed by electrophoresis through agarose.

Taken together, these results indicate that the HBe-retrogen fusion genewas efficiently transduced into murine bone marrow cells and wasexpressed in bone-marrow-derive DCs. Interestingly, the surfaceexpression of MHC-II and costimulation molecules on DCs comprisingtransduced HBe-retrogen were significantly higher than the expression ofthe molecules on DCs transduced with HBeAg or HBcAg. This observationsuggests that binding of the Fc to receptor activated the DCs.

EXAMPLE 3 In Vitro Activation of Naive CD4+-T-cells

To evaluate whether the transduced DCs were capable of priming naiveCD4+-T-cells in cell culture, naive CD4+-T-cells isolated from C57BL/B6mouse spleen cells were co-cultured with murine DCs transduced with theretroviral vectors of Example 1 at a ratio of 1:20 (DCs:T-Cells). CD4+ Tcells were isolated from the suspension of mouse spleens using a CD4+ Tcell enriching column (R&D Systems, Minneapolis, Minn.). CD8+ T cellswere isolated from the suspension of mouse spleens using a CD8+ T cellenriching column (R&D System, Minneapolis, Minn.). Purified CD4+ or CD8+T cells were cultured in RPMI-1640 supplemented with 10% FBS at 37° C.and 5% CO₂.

When naive CD4+-T-cells were co-cultured with the DCs transduced witheither HBcAg, HBeAg or Fc fragment gene, only low or background levelsof granulocyte-macrophage colony-stimulating factor (GM-CSF) andinterferon (IFN)-γ were detected by ELISA in the culture medium.Further, no apparent T-cell proliferation was observed when either cellnumbers or the incorporation rate of ³H-thymidine was monitored. Incontrast, when naive CD4+ T-cells were co-cultured with the DCstransduced with the HBe-retrogen for 5 days, T-cells activelyproliferated and high levels of GM-CSF and IFN-γ were detected in theculture medium (FIGS. 6A and 6B). These results suggest that secretoryHBeAg or cytosolic HBcAg could not be efficiently processed andpresented to MHC-II by DCs. In contrast, secretory HBe-retrogens couldbe efficiently processed following Fc-receptor-mediated internalizationand presentation to MHC-II in DCs, leading to the vitro activation ofnaive CD4+ T-cells.

No apparent naive CD8+ T-cell activation was detected in the co-culturewith the transduced DCs. The failure to detect naive CD8+ T-cellactivation in the cell-culture may be due to the fact that there is onlyone known MHC-I restricted epitope in the HBe Ag and that CD4+-T-cellsare required for efficient activation of CD8+ T-cells (Ridge et al.,1998).

To further demonstrate MHC-II-restricted antigen presentation using theretrogen strategy, MHC-II knockout (KO) C57BL/6 mice, in which MHC-IIantigen presentation by DCs was abolished, were used (Charles River,N.Y.). DCs derived from wild-type (WT) or MHC-II KO mice were transducedwith the HBe-retrogen and were then co-cultured with CD4+ T-cellsobtained from WT mice at a ratio of 1:20. As shown in FIGS. 7A and 7B,only low levels of GM-CSF and IFN-γ were detected in the medium of theco-culture containing the transduced KO-DCs, and no apparent T-cellproliferation was observed. In contrast, when CD4+ T-cells wereco-cultured with the transduced WT DCs for 5 days, the T-cells activelyproliferated and high levels of GM-CSF and IFN-γ in the culture mediumwere detected (FIGS. 7A and 7B).

EXAMPLE 4 In vivo Induction of Helper and Cytotoxic T-cell, and B-cellImmune Response

The potential of the retrogen antigen presentation strategy wasevaluated in vivo. Mice (C57BL/B6) were divided into four groups (4 to 6mice/group) and each mouse was administered about a half a million ofthe DCs that were transduced with HBcAg, HBeAg, Fc, or HBe-retrogen in0.2 ml PBS containing 50,000 U IL-2 (Chiron Corp. Emmeryville, Calif.)by intraperitoneal injection. At the different times post-finaladministration, the mice were sacrificed and peripheral blood, spleensand other organs were collected. T-cells were isolated for analysisusing the CD4 or CD8+ T-cell enriching columns (R&D System, Minneapolis,Minn.).

Three months after the first injection, mice were sacrificed and theperipheral blood, spleen, and other tissue samples were collected. Fromgross examination, the lymph nodes in the peritoneal cavity weresignificantly enlarged in the mice administrated theHBe-retrogen-transduced DCs, while in normal mice and mice administratedother constructs, the lymph nodes were too small to be visible.Histologic examination also revealed active proliferation of T-cells andB-cells in the peritoneal lymph nodes of mice administered theHBe-retrogen-DCs.

EXAMPLE 5 Induction of T_(H)1 and T_(H)2 Helper T-Cells

Mice immunized as in Example 4 were used to determine the induction ofT_(H)1 and T_(H)2 helper T-cells. Skilled artisans are cognizant of theimportance of determining the induction of T_(H)1 and T_(H)2 cells. Itis well known that CD4+-T-cells perform the following functions: 1) theyhelp B-cells develop into antibody producing plasma cells; 2) they helpCD8+-T-cells to become activated cytotoxic T-cells; and 3) they effectdelayed hypersensitivity. These functions are performed by twosubpopulations of CD4 cells: T_(H)1 cells mediate delayedhypersensitivity and produce primarily IL-2 and gamma interferon(IFN-γ), whereas T_(H)2 cells perform the B-cell helper function andprimarily produce IL-4 and IL-5.

CD4+-T-cells were isolated from the spleens of the immunized mice usingan anti-CD4 column (R&D Systems, Minneapolis, Minn.) and these cellswere then co-cultured with DCs of mice that were pulsed with arecombinant HBeAg protein. After only 2 days in co-culture of cellshaving a ratio of T-cells:DCs of 1000:1, the CD4+-T-cells from miceadministered the HBe-retrogen-DCS actively proliferated. High levels ofGM-CSF and IFN-γ (stimulate macrophages and CD8+-T-cells), as well asIL-4 and IL5 (stimulate B-cells), were detected in the co-culturemedium. Anti-CD4 antibodies, but not anti-CD8 antibodies, dramaticallyblocked cytokine production in these co-cultured T-cells. In contrast,when the CD4+-T-cells obtained from mice immunized with HBeAg-, HBcAg-or Fc-DCs were co-cultured with HBeAg-DCs, only low levels of GM-CSF,IFN-γ, IL-4, and IL-5 were detected in the co-culture medium, and noactive T-cell proliferation was observed. Since IL-4 and IL-5 are mainlyproduced by T_(H)2 and GM-CSF and IFN-γ by T_(H)1 cells, the resultsdemonstrate that HBe-retrogen-transduced DCs effectively activate bothT_(H)1 and T_(H)2 T-cells.

EXAMPLE 6 Induction of High Titers of Anti-HBeAg Antibodies

Mice were immunized as in Example 4 to determine the level ofantibodies. Immunization of mice with HBe-retrogen-transduced DCsinduced high-titer, long-lasting anti-HBe/cAg antibody responses inmice. As shown in FIG. 8, significantly higher titers of anti-HBeAgantibodies were detected in the sera of the mice administered theHBe-retrogen-transduced DCs than in the mice administeredHBeAg-transduced DCs. The levels of anti-HBcAg antibodies in the sera ofimmunized mice were assessed using an ELISA. Briefly, microtitre platescoated with HBcAg recombinant proteins (50 ng/well) were incubated withserially diluted sera in a blocking buffer at 4° C. for 2 hours. Boundantibody was detected after incubation with peroxidase-conjugatedantibodies to mouse IgG diluted in blocking buffer. A polyclonalanti-HBcAg antibody obtained from Chiron Corp. (Emmergyville, Calif.)was used as positive control, and normal mouse sera was used as anegative control. The antibody titer was defined as the highest dilutionhaving an OD₄₅₀ value, which was two times above the negative level.

The fold increase of the antibody production observed may be due to thestronger activation of CD4+ helper T-cells. The significantly lowerlevels of anti-HBe/cAg antibodies in the sera of the mice immunized withHBcAg-transduced DCs may be due to the cytosolic location of HBcAg andlack of CD4+ T-cell activation. Thus, the HBe-retrogen is significantlysuperior to other HBeAg and HBcAg constructs for the induction of anantibody response in mammals immunized with the same. Taken together,the results of the mouse model study demonstrate that DCs transducedwith HBe-retrogen induced vigorous CD4+- and CD8+-T-cell activation, aswell as, B-cell activation.

EXAMPLE 7 Vector Construction of an Intracellular Tumor Antigen

MAGE-3 is a cytosolic and nuclear protein lacking a targeting sequencefor the endogenous MHC-II presentation pathway, which makes itspresentation on MHC-II unlikely or difficult. Since there is no mousehomolog, a human MAGE-3 gene was linked to a signal leader sequencederived from a human chemokine RANTES gene to allow the secretion ofMAGE-3. A plasmid encoding the full-length MAGE-3 gene was used as atemplate to amplify the MAGE-3 DNA with a pair of primers: 5′-primer(A): (SEQ. ID. No. 1) 5′-ACGCGTCGACATGCCTCTTGAGCAGAGGAGTCAG-3′,corresponding to the polynucleotide sequence 1 to 24 of the MAGE-3 genewith an additional Sal I restriction site, and 3′-primer (B): (SEQ. ID.No. 2) 5′-CCGCTCGAGTCACTCTTCCCCCTCTCTCAAAAC-3′, correspond-ing to thepolynucleotide sequence 921 to 945 of the MAGE-3 with a Xho I site. Theaddition of the signal leader sequence derived from the human RANTESgene was generated by PCR amplification with a pair of primers:5′-primer (C): (SEQ. ID. No. 3)5′-ACGCGTCGACATGAAGGTCTCCGCGGCAGCCCTCGCTGTCATCCTCATTGCTACTGCCCTCTGCGCTCCTGCATCTGCCATGCCTCTT GAGCAGAGGAGTCAG-3′,corresponding to the RANTES signal leader sequence and to thepolynucleotide sequence 1 to 24 of the MAGE-3 gene with a Sal I site,and 3′-Primer-B. The signal-MAGE-3 fragment (s-MAGE-3) without the stopcodon was generated by PCR with 5′-Primer-C and 3′-primer (D): (SEQ. ID.No. 4) 5′-ATAAGAATGCGGCCGCTCTCTTCCCCCTCTC TCAAAAC-3′, corresponding tothe polynucleotide sequence 921 to 942 of the MAGE-3 with a Not I site).DCs, the most potent APCs, express IgG Fc receptors (FcγRs), whichmediate a privileged antigen internalization route for efficient MHC-IIas well as MHC-I-restricted antigen. Hence, a Fc fragment cDNA derivedfrom a human IgG1a that can efficiently bind to Fc receptors on murineDCs was fused in frame with the modified MAGE-3 gene to mediate MAGE-3internalization by DCs (FIG. 9A). The secretory MAGE-3 fusion gene(s-MAGE-3-Fc) was then cloned into a murine retroviral vector pFB-Neo(Stratagene) (FIG. 9A). The human IgG cDNA Fc fragment was generated byPCR amplification with the plasmid pEE6/CLL-1 containing human IgG1aheavy chain cDNA as a template. The pair of primers for the PCR reactionare: 5′-primer (E), (SEQ. ID. No. 5) 5′-ATAAGCGGCCGCTAAAACTCACACATGCCCA-3′, corresponding to the polynucleotide sequence 785 to 802 ofthe heavy chain with an additional Not I site, and 3′-primer (F), (SEQ.ID. No. 6) 5′-CCGCTCGAGTCATTTACCCGGAGACAGGGAGAG-3′, corresponding to thepolynucleotide sequence 1447 to 1468 of the heavy chain with a Xho Isite. A murine retroviral vector, pFB-Neo (Stratagene), was used forthis study. The retroviral vector s-MAGE-3-Fc was constructed by athree-piece ligation of the s-MAGE-3 fragment without the stop codon,Fc, and Sal I Xho I-cut pFB-Neo. The retroviral vector s-MAGE-3 orMAGE-3 was constructed by inserting the s-MAGE-3 or MAGE-3 gene into SalI/Xho I-cut pFB-Neo, respectively. To construct the IgG Fc expressionvector, the human IgG Fc cDNA fragment was linked with an immunoglobulinheavy chain (VH) signal leader sequence by two PCR reactions. In thefirst PCR reaction, the IgG Fc cDNA was used as a template for theamplification with a pair of primers: 5′ primer, (SEQ. ID. No. 7)5′GCAGCTCCCAGATGGGTCCTGTCCAAAACTCACACATGCCCACCGTG CCCAGCAC-3′,corresponding to the polynucleotide sequence 785 to 815 of the heavychain and a partial VH-leader sequence, and 3′-Primer F (SEQ. ID. No.6). The second PCR utilizing the product of the first PCR as a templatewas carried out with a pair of primers: 5′ primer, (SEQ. ID. No. 8)5′-ACGCGTCGACATG GGAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCC-3′, corresponding to the N-terminal polynucleotide sequence ofthe VH-secretion signal leader sequence with an additional Sal I site,and 3′-Primer F (SEQ. ID. No. 6). The Fc cDNA with a signal leadersequence was then cloned into the retroviral vector. The expressionvector pcDNA3.1-MAGE-3 was constructed by inserting the MAGE-3 into theXhoI/XbaI-cut pcDNA3.1 (Invitrogen). Several control retroviral vectorsexpressing a native, intracellular MAGE-3, secretory s-MAGE-3, orsecretory Fc fragment were also constructed (FIG. 9A). Each resultantvector was identified by restriction enzyme analysis and confirmed byDNA sequencing.

EXAMPLE 8 Production of Retroviruses and Transduction of BoneMarrow-Derived DC

Retroviral vectors were produced by transient transfection. Packagingcells (PA317) were cultured in 100-mm culture dishes with DMEMcontaining 10% heat-inactivated FBS (Gibco-BRL) and transfected with10-15 μg of retroviral vector plasmids (from Example 7, i.e.,intracellular MAGE-3, secretory s-MAGE-3, or secretory Fc fragment) thatwere prepared by using endotoxin-free QIAGEN kits by Lipofectin(Gibco-BRL). After overnight incubation, the medium was replaced withDMEM containing 5% FBS. After 48 hours, the culture medium containingrecombinant retroviruses was harvested and filtered (0.22 μm), asdescribed previously (Chen et al., 1997). To generate DCs, bone marrowcells were flushed from the bones of mouse limbs, passed through a nylonmesh, and depleted of red cells with ammonium chloride. After extensivewashing with RPMI-1640, the cells were incubated with rabbit complements(Calbiochem) and a cocktail of monoclonal antibodies consisting ofanti-CD4, anti-CD8, anti-CD45R/B220, and anti-MHC-II (PharMingen andBioSource International) in RPMI-1640 at 37° C. for 40-60 min. Afterextensive washing with RPMI-1640, cells (5×10⁵ cells/ml) in RPMI-1640supplemented with 6% FBS, 80 ng mSCF/ml (R&D Systems), and 20 Units (U)mIL-6/ml (BioSource International) were plated in 12-well culture plates(2.5 ml/well), incubated at 37° C., 5% CO₂ overnight, and then refedwith fresh medium. After 48-hour incubation, the cells were spun down,resuspended in 1.5 ml of the retrovirus supernatants, placed onto24-well culture plates coated with Retronectin (PanVera) at aconcentration of 10-20 ng/ml, and incubated at 37° C., 5% CO₂ for 3-4hour. The supernatants were then replaced with 1.5 ml of RPMI-1640supplemented with 5% FBS, 10 ng murine stem cell factor (mSCF)/ml, 60 ngmGM-CSF/ml (BioSource International) and 100 U mIL-4/ml (R & D Systems)overnight. The transduction procedure was repeated 2-3 times and about30% of BM cells were usually transduced by this procedure. After thefinal transduction, the cells were washed and cultured in Opti-MEM(Gibco-BRL) containing mGM-CSF and mIL-4 for several days to allowfurther DC differentiation. DCs were further enriched with a 50%FCS-RPMI-1640 sedimentation procedure, as described previously (Inaba etal., 1992).

After several days of culture, a substantial fraction of the cellsshowed distinct DC morphology. The s-MAGE-3-Fc, s-MAGE-3, MAGE-3, or Fcgene in the transduced DCs was transcribed, as demonstrated by reversetranscription (RT)-PCR assays. Quantitative Western blotting analysiswas used to demonstrate protein expression and secretion by theconstructs in transduced DCs. Briefly, the transduced DCs were lysedwith a buffer (Boehringer Mannheim) (10 mM Tris 150 mM NaCl (pH 7.4),1%TX-100 (Sigma), 0.5 mM PMSF, and protease inhibitor cocktail tablets)on ice for 10 min. Cell lysates and culture media were then precipitatedwith a rabbit polyclonal antibody against MAGE-3, followed by incubationwith Protein A-Sepharose (Sigma). The precipitates were then resuspendedin 20 μl loading buffer. The protein samples (20 μl) were loaded onto a10% SDS-PAGE gel and transferred to a Hybond PVDF membrane (AmershamPharmacial Biotech), which was blocked by overnight incubation in PBS(pH 7.5) containing 5% non-fat dried milk (Carnation) and 0.1% (v/v)Tween-20 (Fisher Scientific) at 4° C. After washing with a buffer (PBScontaining 0.1% (v/v) Tween-20), the membrane was incubated with a mousemonoclonal antibody against MAGE-3 diluted in a PBS buffer containing2.5% non-fat milk and 0.1% Tween-20 (1:400) at room temperature for 1hour. After washing, the membrane was then incubated with a horseradishperoxidase (HRP) labeled anti-mouse IgG (Amersham Pharmacia Biotech) inthe buffer (1:10,000) at room temperature for 1 hour. After a finalwash, the membrane was visualized with an ECL-Plus chemiluminescentdetection kit (Amersham Pharmacia Biotech) and exposed on a Kodak film.Protein band intensity of the Western blot on the film was determinedand analyzed by a PhosphorImager (Molecular Dynamics) with anImage-Quant software 1.2 version. It was found that the s-MAGE-3-Fc ands-MAGE-3 proteins were efficiently produced and secreted from DCs, whileMAGE-3 was retained intracellularly (FIGS. 9B and 9C). Comparable levelsof s-MAGE-3-Fc, s-MAGE-3, and MAGE-3 proteins were expressed in thetransduced DCs.

EXAMPLE 9 Interaction of Fc on DC

Interaction of Fc with FcγRs on DCs triggers cell activation, causingthe up-regulation of cell surface molecules involved in antigenpresentation. Surface markers were examined to evaluate whether theexpression of s-MAGE3-Fc in the transduced DCs could induce DCactivation. Surface markers of DCs transduced with s-MAGE-3-Fc,s-MAGE-3, or vector, were measured by flow cytometric assays. Briefly,the DCs were pre-incubated with an anti-CD16/CD32 antibody (2.4G2,PharMingen) for blocking FcγRs at 4° C. for 30-60 min. The DCs were thenincubated with primary antibodies at 4° C. for 30 min, followed byincubation with an anti-mouse or -rabbit IgG-FITC conjugate. Afterextensive washing, the DCs were analyzed by a FACScan (Becton Dickinson)with CellQuest software. As shown in FIGS. 9D, 9E and 9F, higher levelsof MHC class-II, CD40, and CD86 were expressed on DCs derived from BMcells transduced with s-MAGE-3-Fc and on DCs in the presence of LPS thanon DCs transduced with s-MAGE-3 or vector control. These results suggestthat the secretion and subsequent interaction of the fusion protein Fcwith FcγR activate DCs.

EXAMPLE 10 Induction of Potent T_(H)1 In Vivo

To evaluate whether the secretion and subsequent internalization ofMAGE-3 can enhance the immunogenicity of this antigen in vivo, DCs weretransduced with s-MAGE-3-Fc, s-MAGE-3, MAGE-3, or Fc by retroviralvectors, and then administered i.v. once into C57BL/6 mice (0.5-1×10⁵ DCin 30 μl PBS containing 50,000 U IL2(chiron) per mouse). Four to sixweeks after immunization, the mice were sacrificed and peripheralbloods, spleens, and other tissue samples were collected. Lymph nodeswere substantially enlarged in the mice immunized with s-MAGE-3-Fc-DCs,reminiscent of pathogen infection, but not in the mice administered withDCs transduced with s-MAGE-3, MAGE-3, or Fc.

To determine if immunization with transduced DCs can induce CD4+ helperT cell responses, CD4+ T-cells from splenocytes of the immunized micewere isolated and then co-cultured them with bone-marrow (BM)-derivedDCs transduced with s-MAGE-3-Fc. Briefly, CD4+ or CD8+ T-cells wereisolated from spleen suspensions with CD4+ or CD8+ T cell enrichmentcolumns (R & D Systems) and then cultured in RPMI-1640 supplemented with10% FBS for 24 to 48 hours before further analysis. Draining lymph nodesfrom immunized mice were digested with a cocktail of 0.1% DNase I(fraction IX, Sigma) and 1 mg/ml collagenase (Roche MolecularBiochemicals) at 37° C. for 40-60 min. DCs were positively isolated fromthe cell suspensions of lymph nodes or spleens with anti-CD11c (N418)Micro-Beads (Miltenyi Biotec Inc) for further study. During two weeks ofco-culture with different ratios of CD4+ T-cells vs DCs, the CD4+T-cells from mice immunized with s-MAGE-3-DCs, MAGE-3-DCs, or Fc-DCs didnot actively proliferate, and only low levels of IL-2, IFN-γ, TNF-α, andIL-4 were detected in the co-culture media (FIGS. 10A, 10B, 10C and10D). CD4+ T cells from immunized mice were co-cultured with DCs at arate of 1000:1 (T cell:DC, 2×10⁵:2×10²) for various times. Supernatantsof the co-cultures were harvested and subsequently assayed for cytokineconcentrations by ELISA (PharMingen) according to the manufacturer'sinstructions (PharMingen). In contrast, in the co-cultures with CD4+T-cells from mice immunized with s-MAGE-3-Fc-DCs, high levels of IL-2and IFN-γ were detected in the co-culture media after only 48-hour ofco-culture even at a 1:1000 (DC:T-cell) ratio. Anti-CD4, but notanti-CD8 antibodies, blocked the cytokine production by the co-culturedcells (FIG. 11A). Repeated experiments showed similar results. Tofurther determine the specificity of the T-cell responses, BM-derivedDCs transduced with a retroviral vector expressing an irrelevanthepatitis B virus core antigen (HBcAg) were co-cultured with CD4+ Tcells from s-MAGE-3-Fc-DCs-immunized mice. Only low levels of IFN-γ, andother cytokines were detected in the co-culture media (FIG. 11B).Furthermore, DCs from the lymph nodes of mice six weeks afterimmunization were isolated with anti-CD11c microbeads (Miltenyi Biotec,Inc.) and co-cultured with CD4+ T cells from the same immunized mice. Asshown in FIGS. 12A, 12B, 12C, and 12D, high levels of IL-2, IFN-γ, andTNF-α were only detected in the co-cultures of the cells froms-MAGE-3-Fc-DCs-immunized mice. These results indicate that the DCstransduced with s-MAGE-3-Fc can home to lymphoid organs or tissues, andactivate Th1 responses more efficiently than do DCs transduced with thenative MAGE-3 or s-MAGE-3.

EXAMPLE 11 Induction of CTLs In Vivo

The JAM or “just another method” test was performed to determine whetherimmunization with s-MAGE-3-Fc-DCs can induce strong CTL responses. TheJAM test was used to measure cytotoxic activities. Briefly, mice weresacrificed at different times after immunization and a single-cellsuspension of splenocytes was cultured in RPMI 1640 10% FBS. A total of4×10⁶ splenocytes was restimulated with 8×10⁴ γ-irradiated (10,000 rad)syngeneic EL4-MAGE-3 cells or EL4-HBcAg cells/2 ml in 24-well plates(Costar) for 4-6 days in 5% CO₂ at 37° C., pooled, and then resuspendedto 1×10⁷ cells/ml. To label the target cells, ³H-thymidine was addedinto 5×10⁵/ml EL4-MAGE-3 or EL4-HBcAg cells at a final concentration of2 μCi/ml. After 6 hour incubation, the cells were gently washed oncewith PBS and resuspended in the culture medium (1×10⁵ cells/ml).Different numbers of effector cells were then co-cultured with aconstant number of target cells (1×10⁴/well) in 96-well round-bottomedplates (200 μl/well) for 4 hour at 37° C., after which the cells andtheir media were then aspirated onto fiber glass filters (Filter MateHarvester, Packard) that were then extensively washed with water. Afterthe filters were dried and placed onto 96-well plates, 25 μl MicroScint20 (Packard) were added to each well. The plates were counted in aTopCount NXT Microplate Scintillation and Luminescence Counter(Packard). In some experiments, the restimulated effector cellpopulations were incubated with the anti-CD4 or anti-CD8 antibodies (30μl/well, PharMingen) for 30-60 min to deplete CD4+ or CD8+ T-cellsbefore cytotoxicity assays. The percent of specific killing was definedas: (Target cell DNA retained in the absence of T-cells(spontaneous)−Target cell DNA retained in the presence ofT-cells)/spontaneous DNA retained×100. The value of total ³H-thymidineincorporation is often similar to the spontaneous retention. Splenocytesfrom immunized mice were restimulated in vitro in RPMI-1640, 10% FBSwith syngeneic cells EL4-MAGE-3, and then co-cultivated with³H-thymidine labeled EL4-MAGE-3 cells at various effector/target ratiosto measure the specific killing. EL4-MAGE-3 cells were established bytransfection with the MAGE-3 expression vector (pcDNA3.1-MAGE-3) andZeocin (Invitrogen) selection, and shown to express MAGE-3 by PCR andimmunoprecipitation assays.

Splenocytes from mice immunized with s-MAGE-3-Fc-DCs killed target cellsmuch more efficiently than those from mice immunized with s-MAGE-3,MAGE-3, or Fc (FIG. 13). The specificity of killing was furtherdemonstrated by the inability of the splenocytes ofs-MAGE-3-Fc-DCs-immunized mice to kill EL4-HBcAg cells that express theirrelevant HBcAg (FIG. 13), and by the inhibition of killing with theanti-CD8, but not the anti-CD4 antibody. Thus, these results demonstratethe superior ability of s-MAGE-3-Fc-DCs to induce CTL responses, due tothe enhanced T_(H)1 and cross-priming of receptor-mediated antigeninternalization.

EXAMPLE 12 Induction of Antibody

Since antibodies can also play a role in antitumor immunity, anti-MAGE-3antibody titers in the sera of immunized mice (similar to Example 10)were measured by ELISA. Anti-MAGE-3 antibodies in the sera of immunizedmice were detected by ELISA. Briefly, microtiter plates (Dynatech)coated with a recombinant MAGE-3 proteins (50 ng each/well) wereincubated with serially diluted sera in a blocking buffer (KPL,Gaithersburg, Md.) at room temperature for 2 hour. Bound antibody wasdetected after incubation with a peroxidase-conjugated antibody againstmouse IgG (Sigma) diluted in the blocking buffer. A monoclonal antibodyagainst MAGE-3 was used as a positive control and normal mouse sera as anegative control. The antibody titer was defined as the highest dilutionwith an OD_(A450) greater than 0.2. The background OD_(A450) of normalmouse sera was lower than 0.1. Anti-MAGE-3 antibodies were induced 2weeks after DC immunization and reached the peak 4-6 weeks afterimmunization.

As shown in FIG. 14, significantly higher titers of anti-MAGE-3antibodies were detected in the sera of s-MAGE-3-Fc-DC immunized micethan in mice immunized with s-MAGE-3-DCs or MAGE-3-DCs. The specificityof the antibody responses was demonstrated by the lack of antibodyagainst the irrelevant HBcAg in the immunized mice. Taken together, thefindings indicate that s-MAGE-3-Fc-DCs are superior to MAGE-3-DCs ors-MAGE-3-DCs in inducing CD4+ Th, CD8+ CTL, as well as B-cell responses.

EXAMPLE 13 Enhanced Interaction of Helper T-Cells

Primed CD4+ T_(H) cells that recognize their specific peptides in thecontext of MHC-II on DCs greatly increase their interaction withconditioned DCs. This interaction via CD40-CD40L can trigger DCproduction of IL-12 and is critical for generating T-cell helper for CTLresponses. To test if this approach can enhance CD4+ T_(H) interactionwith s-MAGE-3-Fc-DCs, IL-12 production by transduced DCs in co-culturewith primed CD4+ T-cells was measured. Primed CD4+ T-cells were isolatedfrom mice immunized with s-MAGE-3-Fc-DCs and then co-cultured withBM-derived DCs transduced with s-MAGE-3-Fc, s-MAGE-3, MAGE-3, or Fc. Asshown in FIG. 15, a significant increase in IL-12 production wasobserved in the CD4+ T-cell co-culture with s-MAGE-3-Fc-DCs, but not inthe co-cultures with s-MAGE-3-DCs or MAGE-3-DCs. The IL-12 production bys-MAGE-3-Fc-DCs was inhibited by blocking with CD40L on the primed CD4+T-cells. The expression of Fc in DCs also non-specifically enhancedIL-12 production to a lesser degree. These results, together with the invivo results of Example 10, 11, and 12 data, indicate that the secretionand subsequent FcγRs-mediated internalization of MAGE-3 lead to thecross-presentation of MAGE-3 on DCs for the induction of T_(H)1 and CTLresponses.

EXAMPLE 14 Protective Immunity Induced by s-MAGE-3-Fc-DCs

To examine if the enhanced anti-MAGE-3 immune responses could lead toeffective anti-tumor immunity, challenge experiments were performed. TheEL4-MAGE-3 cell line was derived from the parental tumor EL-4 line thatgrows rapidly in syngeneic mice and used for challenge experiments. Whenintradermally implanted into syngeneic C57BL/6 mice, EL4-MAGE-3 cells(0.5 to 1×10⁶ cells) showed aggressive tumor growth similar to that ofparental EL-4 cells, producing visible tumors in mice by only 3-5 daysafter inoculation and resulting in mouse death usually within one monthafter inoculation. To test the ability of s-MAGE-3-Fc-DCs to inhibitEL4-MAGE-3 tumor growth, mice were immunized i.v. twice (7 day interval)with 1×10⁵ DCs tranduced with s-MAGE-3-Fc, s-MAGE-3, MAGE-3 or Fc,followed by challenge with the EL4-MAGE-3 cells (1×10⁶). C57BL/6 micewere immunized by i.v. injection with 1×10⁵ transduced DCs on day 0 andday 7, and then intradermally challenged with 1×10⁶ exponentiallygrowing EL4-MAGE-3 or EL4-HBcAg cells 1 week after the secondimmunization. Tumor sizes were measured every 2 to 3 days, with tumorvolumes calculated as follows: (longest diameter)×(shortest diameter)².

As shown in FIG. 16A, tumor growth was inhibited to a much greaterextent in mice immunized with s-MAGE-3-Fc-DCs, although immunizationwith s-MAGE-3-DCs, MAGE-3-DCs, or even Fc-DCs (a non-specific immunestimulator) did confer some degree of protection. The potency of theantitumor activity shown by these constructs correlated with theirabilities to induce immune responses. Consistently, the mice immunizedwith s-MAGE-3-Fc-DCs survived considerably longer than mice immunizedwith other vector-transduced DCs (FIG. 16B). The antitumor activityinduced by the s-MAGE-3-Fc-DCs was specific, since mice immunized withs-MAGE-3-Fc-DCs and challenged with wild type EL4 or EL4-HBcAg cellsalso developed lethal tumors and died within one month. S-MAGE-3-Fc-DCsalso partially inhibited the growth of established EL4-MAGE-3 tumors inmice, even though the immune system may not have sufficient responsetime to effectively control rapidly lethal tumor growth in this model.

EXAMPLE 15 Construction of an HBe Antigen in a Mammalian ExpressionVector

A plasmid encoding the full-length HBV (adw subtype) genome was obtainedfrom the American Type Culture Collection (ATCC). The HBV precore/coregene was found to contain a single base pair deletion, which causes aframeshift at codon 79, resulting in two consecutive stop codons at 84and 85. This gene was repaired by inserting the deleted base using PCRmutagenesis and confirmed by DNA sequencing. The full-length HBeAg genewas generated by PCR amplification of the repaired HBV genome with apair of primers (5′-primer (P-A): (SEQ. ID. No. 9)5′-TTAAGCTTATGCAACTTTTTCACCTCTGCCTAATC-3′, corresponding to thepolynucleotide sequence 1904 to 2020 of the HBV genome with anadditional HindIII restriction site, and 3′-primer (P-B): (SEQ. ID. No.10) 5′-TTTCTAGAATCGATTAACATTGAGATTCCCGAGA-3′, corresponding to thepolynucleotide sequence 2437 to 2457 of the HBV genome with additionalXba I and Cla I sites). The truncated HBeAg gene with the deletion ofthe arginine-rich, C′-terminal sequence of HBeAg (aa 150-185) that iscleaved during viral infection, was generated by PCR amplification witha pair of primers (5′-primer: P-A (SEQ. ID. No. 9) and 3′-primer (SEQ.ID. No. 11) 5′-GTGCGGCFCGCTCTAACAACAGTAGTTTCCGGAAGTGT-3′, correspondingto the polynucleotide sequence 2324 to 2350 of the HBV genome with anadditional Not I restriction site). The full-length HBcAg gene wasgenerated by PCR amplification with a pair of primers (5′-primer: (SEQ.ID. No. 12) 5′-TTAAGCTTATGGACATTGACCCTTATAAAGAATTTGGAGC-3′,corresponding to the polynucleotide sequence 1901 to 1932 of the HBVgenome with an additional Hind III restriction site, and the primer P-B(SEQ. ID. No. 10)). The human IgG cDNA Fc fragment was generated by PCRamplification with the plasmid pEE6/CLL-1 containing human IgG heavychain cDNA as a template. The pair of primers for the PCR reaction are:5′-primer (SEQ. ID. No. 13) 5′-ATAAGCGGCCGCTAAAACTCACACATGCCCA-3′,corresponding to the polynucleotide sequence 785 to 802 of the heavychain with an additional Not I site, and 3′-primer (P-C) (SEQ. ID. No.14) 5′-TATTCTA GATCGATCACTCATTTACCCGGAGACAGG-3′, corresponding to thepolynucleotide sequence 1447 to 1468 of the heavy chain with a Cia Isite. pRc/CMV vector (Invitrogen) was used for this study. Theexpression vector HBe-Fc, which expresses the secretory HBe-Fc fusionprotein consisting of the truncated HBeAg fused in-frame to the IgG Fc,was constructed by a three-piece ligation of the truncated HBe fragment,IgG Fc, and Hind III/Cla I-cut pRc/CMV vector. The expression vectorHBeAg, which expresses a secretory HBeAg protein, was constructed byinserting the HBeAg gene into the HindIII/ClaI cut-pRc/CMV vector. Theexpression vector HBcAg, which expresses a cytosolic HBcAg protein, wasconstructed by inserting the HBcAg gene into the HindIII/ClaIcut-pRc/CMV vector. To construct the IgG Fc expression vector, the humanIgG Fc cDNA fragment was linked with a mouse VH signal leader sequenceby two PCR reactions. In the first PCR reaction, the IgG Fc cDNA wasused as a template for the amplification with a pair of primers (5′primer (SEQ. ID. No. 15), 5′-GCAGCTCCCAGATGGGTCCTGTCCAAAACTCACACATGCCCACCGTGCCCAGCAC-3′, corresponding to the polynucleotide sequence 785to 815 of the heavy chain and a partial VH-leader sequence, and the3′-primer P-C (SEQ. ID. No. 14)). The second PCR utilizing the productof the first PCR as a template was carried out with a pair of primers(5′ primer, (SEQ. ID. No. 16)5′-TTAAGCTTCATATGGGAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCC-3′, corresponding to the N-terminal polynucleotidesequence of the VH-leader sequence with additional HindIII and NdeIsites, and the 3′ primer P-C (SEQ. ID. No. 14)). The Fc cDNA with aleader sequence was cloned into the HindIII/ClaI cut-pRc/CMV vector.These resultant vectors were identified by restriction enzyme analysisand confirmed by DNA sequencing. Plasmids were transformed into E. colistrain (XL-1 blue) and grown from a single colony for 16-20 hours at 37°C. in the presence of 50 ug/ml ampicillin. DNA was isolated using theEndotoxin-free purification kit (Qiagen) according to standard protocol.DNA was resuspended in endotoxin-free PBS (Sigma) at a finalconcentration of 1 mg/ml. The ratio of OD260/280 ranged from 1.8 to 2.0.DNA was stored −200° C. and analyzed by restriction digestion before theday of immunization.

The Fc fragment derived from a human IgG1a was used as a cell-bindingelement to enhance the internalization of the model HBV nucleocapsidprotein, since DCs express IgG Fc receptors (FcγRs), which mediate aprivileged antigen internalization route for efficient MHC-II as well asI-restricted antigen presentation. Although both HBcAg and HBeAg areencoded by the HBV pre-C/C gene, the secretory HBeAg protein isinitiated at a start codon 29 residues upstream of the start codon forHbcAg. The HBeAg was fused in frame with a human IgG1a Fc fragment cDNAgene, and then cloned into the pRc/CMV. The human IgG Fc fragment canefficiently bind to the Fc receptors on mouse APCs. Control vectorscontaining the HBeAg gene (secretory), Fc fragment gene with a secretionsignal leader sequence (secretory), or HBcAg gene (cytosolic) wereconstructed (FIG. 18). Murine marrow-derived DCs were generated. Inbrief, bone marrow stem cells were cultured in RPMI-1640 supplementedwith 6% of FBS, 60 ng mGM-CSF/ml, and 100 U mIL-4/ml for 4 days. DCswere then cultured in medium containing a mixture of the recombinantHBeAg (100 μg/ml) and HBcAg (100 μg/ml) proteins (American ResearchProducts, Boston, Mass.) for an additional 4 days. Pulsed-DCs (PDCs)were washed twice with 1×PBS at 1000 rpm for 5 min and resuspended inRPMI 1640 for further analysis. By using radiolabeling andimmunoprecipitation/SDS-polyacrylamide gel analyses (PAGE), it was foundthat the HBeAg-Fc proteins (HBe-Fc) were efficiently produced andsecreted from transfected cells (FIG. 19A). Both intracellular andsecreted HBe-Fc were directly precipitated by Protein A beads,indicating that the fusion protein retains its binding ability toProtein A.

EXAMPLE 16 Induction of T_(H)1, Helper T-Cells by HBe-Fc DNA Vaccine InVivo

Mice were immunized to evaluate this strategy in vivo. C57BL/6 or BALB/cmice were divided into four groups and each mouse was immunized with onei.m. injection of 100 ug (25-50 μg(μl) per quadricep) HBcAg, HBeAg, Fc,or HBe-Fc DNA. After 2-4 weeks of immunization, the mice were sacrificedand peripheral blood, spleens, and other tissue samples were collected.

First, splenocytes from the mice 2-4 weeks after immunization with DNAvaccines were re-stimulated with the recombinant HBe/cAg proteins for 5days. T-cells were isolated from restimulated splenocytes, and thenassessed by using the ³H-thymidine incorporation assay. As shown in FIG.20, T cells from the mice immunized with HBe-Fc DNA construct or withHBe-Fc DNA vaccine primed T cells actively proliferated. In contrast,the T cells from the mice immunized with HBeAg, HBcAg, or Fc DNA vaccineor HBeAg, HBcAg and Fc DNA vaccine primed T cells did not activelyproliferate.

CD4+ T-cells from the immunized mice were co-cultured with DCs that werepulsed with recombinant HBeAg and HBcAg, similar to Example 5. During 6days of co-culture with different ratios of T-cells vs DCs, CD4+ T-cellsfrom the mice immunized with HBeAg, HBeAg or Fc construct did notactively proliferate, and only low levels of IL-2 and IFN-γ weredetected in the co-culture media (FIG. 21A and FIG. 21B). In contrast,in the co-cultures with the CD4+ T-cells from the mice immunized withHBe-Fc construct, CD4+ T-cells actively proliferated after only 48-hourco-culture even at a 1:1000 (DC:T-cells) ratio. Further, levels of IL-2and IFN-γ in the co-culture media were significantly higher than thosein the co-cultures with the CD4+ T-cells from the mice administered withHBeAg or HBcAg construct (FIG. 21A and FIG. 21B). Anti-CD4, but notanti-CD8 antibodies, dramatically blocked the production of thesecytokines by the co-cultured cells (FIG. 21C and FIG. 21D). In addition,an irrelevant antigen, the recombinant HBsAg protein (American ResearchProduct, Boston, Mass.), was used to pulse DCs in parallel with HBe/cAg.The HBsAg-pulsed DCs were unable to stimulate the CD4+ T-cells of HBe-Fcconstruct immunized mice in the described assay, demonstrating thespecificity of CD4+ T helper 1 cell responses induced by HBe-Fcconstruct immunization. These results indicate that the HBe-Fc constructcan more efficiently activate T_(H)1 than can the HBeAg or HBcAgconstructs. Significant levels of IL-4 were not detected in any of theexperiments. Since IL-2 and IFN-γ are mainly produced by T_(H)1 cells.The results indicate that HBe-Fc construct induces T_(H)1 response.

EXAMPLE 17 Induction of CTLs In Vivo

To determine whether immunization with HBe-Fc construct can induce CTLresponses, a JAM test was performed, similar to Example 11. Splenocytesfrom different immunized mice were restimulated in vitro for 4-6 days inmedium containing synthetic peptide HBcAg13-27 and then co-cultivatedwith ³H-labeled, peptide (HBcAg13-27)-pulsed target cells EL-4 (H-2^(b))and p815 (H-2^(d)) at varied effector/target ratios to measure targetcell killing. As shown in FIG. 22, splenocytes from mice immunized withHBe-Fc construct demonstrated significantly higher target cell killingthan those from mice immunized with HBeAg or HBcAg. The specificity ofthe killing was demonstrated by the inability of the splenocytes to killHBcAg-pulsed p815 target cells with an H-2^(d) background, and theinhibition of the killing by the anti-CD8, but not anti-CD4 antibody.Furthermore, HBsAg was also used to restimulate splenocytes from HBe-Fcconstruct immunized mice, and no significant killing to HBcAg-pulsedtarget cells was observed by the HBsAg-restimulated splenocytes. Thesuperior cytotoxicity response induced by HBe-Fc construct is due to theenhanced T-helper 1 and the direct MHC class-I presentation ofinternalized HBe-Fc fusion protein by DCs.

EXAMPLE 18 Induction of Antibody

To determine whether HBe-Fc-DC immunization can induce antibodyresponses, anti-HBe/cAg antibody titers were measured in the pooled seraof mice immunized with different vectors, similar to Example 6. As shownin FIG. 23, anti-HBe/cAg antibodies were detected in the sera of miceimmunization with HBe-Fc construct. The specificity of the antibodyresponses was demonstrated by the lack of antibody against HBsAg in theimmunized mice. By contrast, significantly lower antibody titers weredetected in mice immunized with HBeAg or HBcAg construct (FIG. 23).Taken together, HBe-Fc DNA is significantly superior to DNAs expressingnative HBeAg or HBcAg in inducing CD4+ T helper 1 and CD8+ cytotoxicT-cell, as well as B-cell responses.

EXAMPLE 19 Systemic Activation of DCs by HBe-Fc DNA Vaccination

To evaluate the possibility of the HBe-Fc or HBeAg proteins beingsecreted from the transduced cells and circulating throughout the bodyto perform antigen presentation, a DC transfer experiment was performed.DCs were isolated from immunized mice and transferred into naive mice toassess whether the transferred DCs can prime naive CD4+ and CD8+T-cells. Mice immunized with the HBeAg-Fc, PEA-HBe, or control DNAvaccine were sacrificed one month later. Mouse CD11c (N418) MicroBeads(Miltenyi Biotec) were used to isolate DCs from mouse spleens. CD11c+DCs were injected (IP or IV) into naive mice (about 1-5×10⁵/mouse). Twoto four weeks after the DC transfer, the mice were sacrificed and theantigen-specific CD4+ and CD8+ T-cell responses of different mice aremonitored. As shown in FIG. 24, DCs from splenocytes of HBe-Fc immunizedmice efficiently activate naive T-cell responses, while DCs fromsplenocytes of HBe or HBc immunized mice failed to activate T-cell innaive mice. This result, together with results of the PCR andinternalization assays, indicate that DC antigen presentation isenhanced by FcγR-mediated antigen endocytosis.

EXAMPLE 20 Secretion of Altered Membrane and Intracellular Proteins

Membrane proteins and intracellular proteins, which contain a sequenceto prevent protein membrane translocation and secretion or lack a signalsequence for secretion, can be used for the strategy of the presentinvention without further modification. It is envisioned that deletionor mutation of the sequence which blocks a protein from secretionresults in protein secretion. Membrane proteins often contain a highproportion of hydrophobic amino acids, thus altering the hydrophobicityof these proteins allows them to be targeted for secretion. One skilledin the art recognizes that the retrogen strategy also can be used toenhance immunogenicity of these proteins. Two examples for the deletionor mutation of membrane proteins are HPV E7 and EBV proteins.

E7 is a cytosolic protein. The presence of a string of charged residueshamper the secretion of the protein. Elimination of these residuesfacilitate the protein secretion and stabilize the protein (FIG. 17).Accordingly, the string of charged residues of HPV 16 E7 proteins wasdeleted in current construct (solid box) by two PCR reactions. As aresult, secretion of the truncated E7 proteins after linking with aleader signal (IL-2) was dramatically enhanced.

EBV nuclear antigen 1 is a nuclear protein, which contains a stretch ofhydrophobic amino acid residues which would interfere with proteinmembrane translocation and secretion. In a study, the stretch ofhydrophobic amino acid residues in the EBNA1 protein was deleted. As aresult, the truncated EBNA1 protein was efficiently secreted from cellsafter linking with a leader signal sequence.

In addition to deletion or truncation of the sequence, one skilled inthe art recognizes that the sequence can also be mutated to reduce thehydrophobicity of the protein. Site-directed mutagenesis provides forthe preparation and testing of sequence variants by introducing one ormore polynucleotide sequence changes into a selected DNA.

In general, one first obtains a single-stranded vector, or melts twostrands of a double-stranded vector, which includes within its sequencea DNA sequence encoding the desired protein or genetic element. Anoligonucleotide primer bearing the desired mutated sequence,synthetically prepared, is then annealed with the single-stranded DNApreparation, taking into account the degree of mismatch when selectinghybridization conditions. The hybridized product is subjected to DNApolymerizing enzymes such as E. coli polymerase I (Klenow fragment) inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed, wherein one strand encodes the originalnon-mutated sequence, and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate hostcells, such as E. coli cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

Site-directed mutagenesis is disclosed in U.S. Pat. Nos. 5,220,007;5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377; and 5,789,166.

In addition to membrane proteins, intracellular proteins are modified,resulting in secretion. One such modification is merely the addition ofa signal leader sequence. For example, MAGE is an intracellular proteinthat lacks a signal sequence for secretion. In Example 7, a signalsequence was added to MAGE by using PCR techniques. The addition of thesignal sequence to MAGE enabled this intracellular protein to besecreted. Another modification of an intracellular protein is to alterthe precursor, which is typically an intracellular protein, so that itis secreted similar to the mature protein. For example, the IL-1 betaprecursor protein is cytosolic, but the mature protein is secreted.Thus, Siders and Mizel (J. Biol. Chem., 1995) truncated amino acidresidues in the precursor protein. They illustrated that deletion of afew amino acids between 100 and 104 increased the secretion level of thetruncated protein to the level of the mature IL-1 betas. Thus, oneskilled in the art would be able to utilize this information to alterother intracellular proteins.

A further modification includes the use of viral particles, which arereleased from cells. Thus, the retrogen is fused to a viral gene andassembled into viral particles for release. A virus particle consists ofa nucleic acid genome surrounded by a shell of protein. Packing of viralparticles is performed by any of the methods well-known in the art.

EXAMPLE 21 Protein Glycosylation

Glycosylation of IgG-F_(c) is known in the art to be essential foroptimal activation of effector cells via F_(c)γR recognition. Thus,recombinant fusion proteins containing the Fc moiety must be generatedin a system capable of gycoslylation if binding to FcγR is essential forits potential utility. The baculovirus-insect cell system is commonlyused to generate high yield recombinant protein. The ability of thissystem to add a core oligosaccharide and outer arm sugar residues toglycoproteins is well known by the skilled artisan and makes it asuitable system for expression and purification of the HBe-Fc fusionprotein.

The 1230 bp HBeFc fragment contained in the tHBeAgFc plasmid, whichexpresses the secretory HBe-Fc protein consisting of the truncated HBeAgin-frame fused to the IgG Fc, was constructed. Briefly, recombinantHBe-Fc baculovirus was generated using the pFastBac system (Gibco BRL)with the pFB1 donor plasmid. The HBe-Fc fragment was first PCR amplifiedfrom tHBeAgFc template using the 5′ primer (SEQ. ID. No. 17)5′-GATCGAATTCATGCAACTTTTTCACCTCTGC-3′ and the 3′ primer (SEQ. ID. NO.18) 5′-GATCAAGCTTTCATTTACCCGGAGACAGGGA-3′ to introduce EcoRI and HindIIIrestriction sites to the 5′ and 3′ ends, respectively. This PCR productwas gel purified, digested, and ligated into EcoRI/HindIII cut pFB1donor plasmid. The resultant vector (pFB1-HBeFc) was identified byrestriction enzyme analysis and confirmed by DNA sequencing.Site-specific transposition of the HBe-Fc expression cassette from thedonor plasmid into the baculovirus genome was performed by transformingDH10Bac E. coli with the pFB1-HBeFc donor plasmid. Recombinantbaculovirus were identified by X-ga1selection, as transposition into thebacmid disrupts expression of the lacZα peptide. Recombinant bacmid DNAwas isolated by mini-prep and used to transfect Sf9 insect cellsaccording to the manufacturers' instructions.

The viral stock obtained from the initial transfection was amplified byinfecting a 50 ml suspension culture of Sf9 cells at 2×10⁶ cells/ml with0.5 ml of the viral stock, and collecting the supernatant after 48hours. This stock was then subjected to two additional rounds ofamplification at which point >90% of cells were producing recombinantHBe-Fc as monitored by immunofluorescent staining of infected cells. Theamplified stock was then used to infect four 100 ml cultures of Sf9cells for 72-90 hours. Supernatants were harvested and clarified bycentrifugation for 20 minutes at 14,000 RPM, 4° C. The clarifiedsupernatant was then passed twice over a 5 ml Detergent Absorber GelColumn (Boehringer Mannheim) to remove pluronic that could interferewith protein recovery. Recombinant HBe-Fc protein was then purified fromthe supenatant by passage over a protein G column (Pharmacia) at a flowrate of 1 ml/minute. The column was washed sequentially with 10 volumesof 100 mM Tris pH 6.0 and 10 mM Tris pH 6.0, and the protein eluted in 1ml fractions with 10 volumes of 100 mM Glycine pH 2.7. The pH of allfractions was immediately adjusted to neutral by addition of 1/10 volume1M Tris pH 8.0. Protein containing fractions were determined by A₂₈₀ andseparated by 12% SDS-PAGE to determine purity. Purified fractions werethen subjected to Western Blot. Briefly, 15 μg of the major proteincontaining eluted fraction was separated by 12% SDS-PAGE under reducing(R) or non-reducing (NR) conditions, transferred to nitrocellulose,blotted, and developed using ECL Western blotting detection reagents.Primary antibody, rabbit anti-HBc; secondary antibody, mouseanti-rabbit-peroxidase conjugate.

EXAMPLE 22 Identifications of MHC-II-Restricted Antigens

The present invention is used to identify MHC-II-restricted viralantigens, HIV, HCV, EBV, bacterial antigens, other pathogen antigens,tumor antigens, and self antigens related to autoimmune diseases. Theexpression vector in the present invention has been modified to include“test” polynucleotides. The polynucleotide sequences that are not knownto elicit an immune response. This strategy of the present inventionidentifies new antigens/epitopes that are used to develop new vaccines.

First, a cDNA library is constructed using mRNA from selected cells,i.e., tumor cells. When cDNA is prepared from cells or tissue thatexpress the polynucleotide sequences of interest at extremely highlevels, the majority of cDNA clones that contain the polynucleotidesequence, which can be selected with minimal effort. For less abundantlytranscribed polynucleotide sequences, various methods can be used toenrich for particular mRNAs before making the library. Retroviruses areused as a vector for the library. Retroviral libraries provide the idealway to deliver a high-complexity library into virtually any mitoticallyactive cell type for expression cloning. Because the viral particlesinfect with high efficiency, they deliver a more complex library thantransfection-based methods. One skilled in the art realizes that anyvector can be used for the library. A cDNA library is constructed byusing methods well known in the art. Briefly, tumor cell lines areestablished from tumor samples. CD4+ T-cells from the same mammalperipheral bloods are expanded by co-culture with the mammal tumorlysate-pulsed DCs derived from monocytes/macrophages. These tumor cellsthat are recognized by expanded autologous CD4+ T-cells are identified.Next, the cell lines are plated in 96 wells. Expanded autologous CD4+T-cells are added into the 96-wells, and the IFN-γ or GM-CSFconcentrations in the 96-well co-cultures are monitored. The next stepis to culture and extract mRNA from the positive tumor cells. Theisolated mRNA is converted to cDNA and inserted into a vector, forexample, lentiviral vector with a GFP marker or the test cDNAs arecloned into the expression vector of the present invention. The testcDNAs are cloned into the vector between the signal sequence and thecellular binding element as depicted, for example, in FIG. 25. Once thecDNA library is constructed, the viral vectors are transfected intopackaging cells. Next, immature DCs derived from monocytes from themammal with the same MHC-II genotype are transduced with the recombinantvectors and efficiency is determined. Transduced DCs are co-culturedwith expanded autologous CD4+ T-cells. Positive clones are identified byELISA (GM-CSF) or IL2 surface expression by flow cytometric array. Thepositive clone is PCR amplified and sequenced to determine the protein(FIG. 26).

The human genome is screened to identify the polynucleotide sequencesthat encode proteins and epitopes that are recognized by CD4+ T-cells.These polynucleotide products are used for cancer therapy or to induceimmune tolerance for autoimmune disease therapy, or gene therapy. Thisbasic screening procedure provides for the identification of epitopesfor designing small therapeutic molecules.

Thus, a skilled artisan is cognizant that this screening procedure ismodified to screen a variety of genomes, i.e., human, viral, bacterial,or parasitic. Construction of cDNA libraries are well known in the art.Thus, a skilled artisan is capable of utilizing this information toalter the present invention to identify antigens.

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objectives and obtain the ends andadvantages mentioned as well as those inherent therein. Vaccines,vectors, methods, procedures and techniques described herein arepresently representative of the preferred embodiments and are intendedto be exemplary and are not intended as limitations of the scope.Changes therein and other uses will occur to those skilled in the artwhich are encompassed within the spirit of the invention or defined bythe scope of the pending claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 19 <210> SEQ ID NO 1 <211> LENGTH: 34<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1acgcgtcgac atgcctcttg agcagaggag tcag        #                  #        34 <210> SEQ ID NO 2 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2ccgctcgagt cactcttccc cctctctcaa aac        #                  #         33 <210> SEQ ID NO 3 <211> LENGTH: 103 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 3acgcgtcgac atgaaggtct ccgcggcagc cctcgctgtc atcctcattg ct#actgccct     60 ctgcgctcct gcatctgcca tgcctcttga gcagaggagt cag    #                   #103 <210> SEQ ID NO 4 <211> LENGTH: 38<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4ataagaatgc ggccgctctc ttccccctct ctcaaaac       #                  #     38 <210> SEQ ID NO 5 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 5ataagcggcc gctaaaactc acacatgccc a         #                  #          31 <210> SEQ ID NO 6 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 6ccgctcgagt catttacccg gagacaggga gag        #                  #         33 <210> SEQ ID NO 7 <211> LENGTH: 55 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7gcagctccca gatgggtcct gtccaaaact cacacatgcc caccgtgccc ag#cac          55 <210> SEQ ID NO 8 <211> LENGTH: 68 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 8acgcgtcgac atgggaacat ctgtggttct tccttctcct ggtggcagct cc#cagatggg     60 tcctgtcc                 #                  #                   #          68 <210> SEQ ID NO 9 <211> LENGTH: 35<212> TYPE: DNA <213> ORGANISM: Hepatitis B virus <400> SEQUENCE: 9ttaagcttat gcaacttttt cacctctgcc taatc        #                  #       35 <210> SEQ ID NO 10 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: Hepatitis B virus <400> SEQUENCE: 10tttctagaat cgattaacat tgagattccc gaga        #                  #        34 <210> SEQ ID NO 11 <211> LENGTH: 37 <212> TYPE: DNA<213> ORGANISM: Hepatitis B virus <400> SEQUENCE: 11gtgcggccgc tctaacaaca gtagtttccg gaagtgt       #                  #      37 <210> SEQ ID NO 12 <211> LENGTH: 40 <212> TYPE: DNA<213> ORGANISM: Hepatitis B virus <400> SEQUENCE: 12ttaagcttat ggacattgac ccttataaag aatttggagc      #                  #    40 <210> SEQ ID NO 13 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 13ataagcggcc gctaaaactc acacatgccc a         #                  #          31 <210> SEQ ID NO 14 <211> LENGTH: 36 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 14tattctagat cgatcactca tttacccgga gacagg       #                  #       36 <210> SEQ ID NO 15 <211> LENGTH: 55 <212> TYPE: DNA<213> ORGANISM: Homo sapiens (first 30)/Murine (la #st 25)<400> SEQUENCE: 15gcagctccca gatgggtcct gtccaaaact cacacatgcc caccgtgccc ag#cac          55 <210> SEQ ID NO 16 <211> LENGTH: 69 <212> TYPE: DNA<213> ORGANISM: Murine <400> SEQUENCE: 16ttaagcttca tatgggaaca tctgtggttc ttccttctcc tggtggcagc tc#ccagatgg     60 gtcctgtcc                 #                  #                   #         69 <210> SEQ ID NO 17 <211> LENGTH: 31<212> TYPE: DNA <213> ORGANISM: Hepatitis B virus <400> SEQUENCE: 17gatcgaattc atgcaacttt ttcacctctg c         #                  #          31 <210> SEQ ID NO 18 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 18gatcaagctt tcatttaccc ggagacaggg a         #                  #          31 <210> SEQ ID NO 19 <211> LENGTH: 98 <212> TYPE: PRT<213> ORGANISM: Human papillomavirus type E7 <400> SEQUENCE: 19Met His Gly Asp Thr Pro Thr Leu His Glu Ty #r Met Leu Asp Leu Gln  1               5  #                 10  #                 15Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gl #n Leu Ser Asp Ser Ser             20      #             25      #             30Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gl #y Gln Ala Glu Pro Asp         35          #         40          #         45Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cy #s Lys Cys Asp Ser Thr     50              #     55              #     60Leu Arg Leu Cys Val Gln Ser Thr His Val As #p Ile Arg Thr Leu Glu 65                  # 70                  # 75                  # 80Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cy #s Pro Ile Cys Ser Gln                 85  #                 90  #                 95 Lys Pro

We claim:
 1. An expression vector comprising a polynucleotide promotersequence, a polynucleotide encoding a signal sequence, a polynucleotideencoding an antigen, a polynucleotide encoding a cell binding element,and a polynucleotide polyadenylation sequence all operatively linked,wherein said polynucleotide encoding an antigen and said polynucleotideencoding a cell binding element are interchangeably linked.
 2. Theexpression vector of claim 1, wherein said polynucleotide encoding anantigen is under the transcriptional control of a promoter, and furtherwherein said antigen is a fusion protein expressed from an internalribosome entry site (IRES) sequence.
 3. The expression vector of claim1, wherein said signal sequence is derived from human chemokine RANTESgene.
 4. The expression vector of claim 1, wherein said polyadenylationsequence is selected from a group of SV40, LTR, and bovine hormonepolyadenylation sequences.
 5. An expression vector comprising apolynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding an antigen, a polynucleotideencoding a cell binding element, and a polynucleotide polyadenylationsequence all operatively linked, wherein said antigen is MAGE-3 or afragment thereof, and wherein said polynucleotide encoding a signalsequence is derived from human chemokine RANTES gene, and furtherwherein said a cell binding element is human IgG1a or a fragmentthereof.
 6. The expression vector of claim 5, wherein said expressionvector is selected from the group of a viral vector, a bacterial vectorand a mammalian vector.
 7. The expression vector of claim 5, whereinsaid vector further comprises an integration signal sequence whichfacilitates integration of said expression vector into the genome of acell.
 8. A cell comprising the expression vector of claim
 5. 9. A fusionprotein comprising an antigen and a cell binding element encoded by anexpression vector, wherein said expression vector comprises apolynucleotide promoter sequence, a polynucleotide encoding a signalsequence, a polynucleotide encoding an antigen, a polynucleotideencoding a cell binding element, and a polynucleotide polyadenylationsequence all operatively linked, wherein said antigen is MAGE-3 or afragment thereof, and wherein said polynucleotide encoding a signalsequence is derived from human chemokine RANTES gene, and furtherwherein said a cell binding element is human IgG1a or a fragmentthereof.
 10. A vaccine comprising the expression vector of claim
 5. 11.A vaccine comprising an antigen presenting cell, wherein said antigenpresenting cell is transfected in vitro with the expression vector ofclaim
 5. 12. The vaccine of claim 11, wherein said antigen presentingcell is a dendritic cell.
 13. A vaccine comprising the fusion protein ofclaim
 9. 14. A vaccine comprising an antigen presenting cell whereinsaid antigen presenting cell is transduced in vitro with the fusionprotein of claim
 9. 15. A vaccine of claim 14 wherein said antigenpresenting cell is a dendritic cell.
 16. A method of producing avaccine, said method comprising the steps of: transfecting an antigenpresenting cell with an expression vector, wherein said expressionvector comprises a polynucleotide promoter sequence, a polynucleotideencoding a signal sequence, a polynucleotide encoding an antigen, apolynucleotide encoding a cell binding element, and a polynucleotidepolyadenylation sequence all operatively linked, wherein said antigen isMAGE-3 or a fragment thereof, and wherein said polynucleotide encoding asignal sequence is derived from human chemokine RANTES gene, and furtherwherein said a cell binding element is human IgG1a or a fragmentthereof; and expressing said vector to produce an antigen underconditions wherein said antigen is secreted from said antigen presentingcell.
 17. The method of claim 16, wherein said antigen presenting cellis a dendritic cell.
 18. A method of eliciting an immune response in amammal directed against an antigen, said method comprising: introducingan expression vector into a first cell, wherein said expression vectorcomprises a polynucleotide promoter sequence, a polynucleotide encodinga signal sequence, a polynucleotide encoding an antigen, apolynucleotide encoding a cell binding element, and a polynucleotidepolyadenylation sequence all operatively linked, wherein said antigen isMAGE-3 or a fragment thereof, and wherein said polynucleotide encoding asignal sequence is derived from human chemokine RANTES gene, and furtherwherein said a cell binding element is human IgG1a or a fragmentthereof; and administering said first cell to said mammal, wherein saidfirst cell expresses said expression vector to produce said antigen,wherein said antigen is secreted from said first cell and is endocytosedinto said first cell or a second cell, wherein said endocytosed antigenis processed in said first cell or second cell, and said processedantigen elicits an immune response in said mammal.
 19. The method ofclaim 18, wherein said first cell is a dendritic cell.
 20. The method ofclaim 18, wherein said immune response is a T-cell mediated immuneresponse.
 21. The method of claim 18, wherein said mammal is a human.